Use of pectin or pectin-related saccharides to enhance efficacy of plant growth-promoting rhizobacteria (pgpr) strains for promoting growth and health in plants and animals

ABSTRACT

Disclosed are compositions and methods that include or utilize plant growth promoting rhizobacteria (PGPR) for improving growth and health in plants and animals. The compositions and methods include or utilize a plant growth promoting rhizobacteria (PGPR) that expresses a protein associated with pectin metabolism, and a saccharide comprising pectin or a pectin-related saccharide.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part (CIP) under 35 U.S.C.§ 365(c) of International Application No. PCT/US2015/053239, filed onSep. 30, 2015, which international application claims the benefit ofpriority under 35 U.S.C. § 119(e) to U.S. provisional application No.62/057,667, filed on Sep. 30, 2014, the content of which applicationsare incorporated herein by reference in their entireties.

FIELD

The presently disclosed subject matter relates to the field of plantgrowth-promoting rhizobacteria (PGPR). In particular, the presentsubject matter relates to the use of pectin or pectin-related saccharideto enhance the efficacy of PGPR in regard to promoting growth and healthin plants and animals.

BACKGROUND

Plant-associated microorganisms have been extensively examined for theirroles in natural and induced suppressiveness of soilborne diseases.Among the many groups of such organisms are root-associated bacteria,which generally represent a subset of soil bacteria. Rhizobacteria are asubset of total rhizosphere bacteria which have the capacity, uponre-introduction to seeds or vegetative plant parts (such as potato seedpieces), to colonize the developing root system in the presence ofcompeting soil microflora. Root colonization is typically examined byquantifying bacterial populations on root surfaces; however, somerhizobacteria can also enter roots and establish at least a limitedendophytic phase. Hence, root colonization may be viewed as a continuumfrom the rhizosphere to the rhizoplane to internal tissues of roots.

Rhizobacteria which exert a beneficial effect on the plant beingcolonized are termed “plant-growth promoting rhizobacteria” or “PGPR.”PGPR may benefit the host by causing plant growth promotion orbiological disease control. The same strain of PGPR may cause bothgrowth promotion and biological control. Among the soilborne pathogensshown to be negatively affected by PGPR are Aphanomyces spp., Fusariumoxysporum, Gaeumannomyces graminis, Phytophthora spp., Pythium spp.,Rhizoctonia solani, Sclerotium rolfsii, Thielaviopsis basicola, andVerticillium spp. In most of these cases, biological control resultsfrom bacterial production of metabolites which directly inhibit thepathogen, such as antibiotics, hydrogen cyanide, iron-chelatingsiderophores, and cell wall-degrading enzymes. Plant growth promotion byPGPR may also be an indirect mechanism of biological control, leading toa reduction in the probability of a plant contracting a disease when thegrowth promotion results in shortening the time that a plant is in asusceptible state, e.g. in the case where PGPR cause enhanced seedlingemergence rate, thereby reducing the susceptible time for pre-emergencedamping-off. An alternative mechanism for biological control by PGPR isinduced systemic resistance. PGPR and uses thereof are disclosed in theprior art. (See, e.g., U.S. Pat. Nos. 8,445,255; 6,524,998; 5,935,839;5,640,803; 5,503,652; and 5,503,651; the contents of which areincorporated herein by reference in their entirety).

In addition to their observed association in nature with plants, PGPRalso may be utilized as probiotics for animals in order to improveanimal growth or animal health. For example, Bacillus amyloliquefacienssubsp. plantarum AP193 has been described as a probiotic for fish. (SeeU.S. Published Application No. 2012/0328572).

In swine, probiotics have been used to have a positive influence on gutmicrobiota balance, intestinal epithelium integrity and maturation ofgut-associated tissue. (See Corcionivoshi et al., Animal Science andBiotechnologies, 2010, 43(1)). In poultry, probiotics have been used tomaintain digestive microbial balance and to reduce potential pathogenicbacteria which results in improving growth, egg production, and feedconversion. (See id.). In cattle, probiotics have been used to preventand combat digestive disorders such as diarrhea during lactation, toinfluence ruminal metabolism of nutrients, which helps maintain healthand improve productive performance. (See id.). In sheep, probiotics havebeen used to prevent and combat pathological conditions that arise fromdigestive balance. (See id.).

Therefore, new compositions and methods of use for PGPR in promotinggrowth and health in plants and animals are desirable.

SUMMARY

Disclosed are compositions and methods that include or utilize plantgrowth promoting rhizobacteria (PGPR) for improving growth and health inplants and animals. The compositions and methods include or utilize aplant growth promoting rhizobacteria (PGPR) that expresses a proteinassociated with pectin metabolism, and a saccharide comprising pectin ora pectin-related saccharide.

The disclosed compositions may include inoculants which comprise: (a) aplant growth promoting rhizobacteria (PGPR) that expresses a proteinassociated with pectin metabolism; and (b) a saccharide comprisingpectin or a pectin-related saccharide. Suitable PGPR may includeBacillus species such as Bacillus amyloliquefaciens subspeciesplantarum. The pectin or pectin-related saccharides may includepectin-derived saccharides such as hydrolyzed pectin, D-galacturonate,D-glucuronate, or mixtures thereof. Optionally, the pectin orpectin-related saccharide functions as a carrier for the PGPR and/or theinoculant includes a carrier other than the pectin or pectin-relatedsaccharide.

The disclosed compositions may be used to treat plants, seeds, and soilsin order to improve plant growth or plant health. The disclosedcompositions may be formulated as a plant treatment composition, acoating for seeds, or a soil amendment composition.

The disclosed compositions also may be administered to animals in orderto improve animal growth or animal health. The disclosed compositionsmay be formulated as an animal feed, such as a pelleted animal feed.

Also disclosed are methods of using pectin or pectin-related saccharidesand PGPR in regard to promoting growth or health in plants and animals.The disclosed methods for improving plant growth or plant health mayinclude: (a) treating plants, seeds, or soil with a plant growthpromoting rhizobacteria (PGPR) that expresses a protein associated withpectin metabolism and (b) treating the plants, seeds, or soil with asaccharide comprising pectin or a pectin-related saccharide, where theplants, seeds, or soil may be treated with the PGPR and the saccharideconcurrently or are treated with the PGPR and saccharide non-currentlyin either order.

The disclosed methods for improving animal growth or animal health mayinclude (a) administering to an animal a plant growth promotingrhizobacteria (PGPR) that expresses a protein associated with pectinmetabolism and (b) administering to the animal a saccharide comprisingpectin or a pectin-related saccharide, where the animals may beadministered the PGPR and the saccharide concurrently or are treatedwith the PGPR and saccharide non-currently in either order.

Also disclosed are methods of using pectin or pectin-related saccharidesand PGPR in regard to promoting and/or increasing nodulation innitrogen-fixing plants such as nitrogen-fixing legumes. The disclosedmethods for promoting and/or increasing nodulation in a legume mayinclude: (a) treating the legume, seeds of the legume, or soilsurrounding the legume with a plant growth promoting rhizobacteria(PGPR) that expresses a protein associated with pectin metabolism and(b) treating the legume, seeds of the legume, or soil surrounding thelegume with a saccharide comprising pectin or a pectin-relatedsaccharide, where the legume, seeds of the legume, or soil surroundingthe legume may be treated with the PGPR and the saccharide concurrentlyor the legume, seeds of the legume, or soil surrounding the legume aretreated with the PGPR and saccharide non-currently in either order.

Also disclosed are methods of using pectin or pectin-related saccharidesto prepare compositions and inoculants as disclosed herein. The methodsmay include combining PGPR and pectin, which has been extracted frompectin-containing plant material, or pectin-related saccharides toprepare the disclosed compositions and inoculants. Optionally, a carriermay be combined with the PGPR and pectin or pectin-related saccharidesto prepare the disclosed compositions and inoculants.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Expression of a pectin lyase activity by PGPR Bap strain AP193.Note the cleared halo around the growth of the Bap strain due to pectindegradation.

FIG. 2. Use of 1% pectin as a sole C source by PGPR strains AP143 andAP193 in TSS medium. The small increase in OD₆₀₀ by the non-PGPR strainHD73 was due to residual nutrients present from the previous TSBculture.

FIG. 3. Effect of PGPR and/or 0.1% (w/w) pectin soil amendment onsoybean root and growth after 4 weeks. By strains AP143 and AP193 wereapplied as seed inoculants with 10⁶ CFU/seed and plants (n=11/treatment)were grown in a greenhouse. Treatment groups assigned different lettersare significantly different (P<0.5).

FIG. 4. A. Representative soybean roots grown in the presence of absenceof 0.1% pectin amended to soil and seed inoculated with 10⁶ CFU Bystrain AP142. B. Effect of treatment groups that include 10⁶ CFU/seed Bystrains, with and without 0.1% pectin, on soybean nodulation (n=11).Treatment groups assigned different letters are significantly different(P<0.5).

DETAILED DESCRIPTION

The disclosed subject matter of the invention may be described usingvarious terms as described below.

Unless otherwise specified or indicated by context, the terms “a”, “an”,and “the” mean “one or more.” For example, “a sugar” should beinterpreted to mean “one or more sugars” unless otherwise specified orindicated by context.

As used herein, “about”, “approximately,” “substantially,” and“significantly” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which they are used.If there are uses of the term which are not clear to persons of ordinaryskill in the art given the context in which it is used, “about” and“approximately” will mean plus or minus ≤10% of the particular term and“substantially” and “significantly” will mean plus or minus >10% of theparticular term.

As used herein, “about”, “approximately,” “substantially,” and“significantly” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which they are used.If there are uses of the term which are not clear to persons of ordinaryskill in the art given the context in which it is used, “about” and“approximately” will mean plus or minus ≤10% of the particular term and“substantially” and “significantly” will mean plus or minus >10% of theparticular term.

As used herein, the terms “include” and “including” have the samemeaning as the terms “comprise” and “comprising.” The terms “comprise”and “comprising” should be interpreted as being “open” transitionalterms that permit the inclusion of additional components further tothose components recited in the claims. The terms “consist” and“consisting of” should be interpreted as being “closed” transitionalterms that do not permit the inclusion of additional components otherthan the components recited in the claims. The term “consistingessentially of” should be interpreted to be partially closed andallowing the inclusion only of additional components that do notfundamentally alter the nature of the claimed subject matter.

The term “plant” as utilized herein should be interpreted broadly andmay include angiosperms and gymnosperms, dicots and monocots, and trees.Examples of angiosperm dicots may include, but are not limited totomato, tobacco, cotton, rapeseed, field beans, soybeans, peppers,lettuce, peas, alfalfa, clover, cabbage, broccoli, cauliflower, brusselsprouts), radish, carrot, beets, eggplant, spinach, cucumber, squash,melons, cantaloupe, and sunflowers. Example of angiosperm monocots mayinclude, but are not limited to asparagus, field and sweet com, barley,wheat, rice, sorghum, onion, pearl millet, rye, oats, and sugar cane.Woody plants may include, but are not limited to fruit trees, acacia,alder, aspen, beech, birch, sweet gum, sycamore, poplar, willow, fir,pine, spruce, larch, cedar, and hemlock.

The term “plant” may include nitrogen-fixing plants such asnitrogen-fixing legumes. As is understood in the art, a “legume” is aplant belonging to the family Facaceae or Leguminosae. Most legumes haveroot nodules comprising symbiotic nitrogen-fixing bacteria. Thesymbiotic nitrogen-fixing bacteria of root nodules are capable of takingatmospheric nitrogen (N₂) and reducing the atmospheric N₂ to ammonia viathe reaction: N₂+8H⁺+8e⁻→2NH₃+H₂. The ammonia thus produced can befurther reduced to ammonium by the following reaction: NH₃+H^(+→NH) ₄ ⁺.The ammonium thus produced can be used by the legume as a nitrogensource for growth. As such, root nodules and the symbioticnitrogen-fixing bacteria therewithin are important for plant growth andmethods and compositions that promote and/or increase nodulation aredesirable.

The term “animal” as utilized herein should be interpreted broadly andmay include mammals and non-mammals. Mammals may include human andnon-human mammals, such as cows, pigs, sheep, and the like. Non-mammalsmay include birds (e.g., chickens, turkeys, ducks, and the like) andfish.

Non-human animals may include aquatic animals. In particular, aquaticanimals may include farmed fish (e.g. catfish or tilapia) andcrustaceans (e.g., shrimp).

The present inventors have identified a collection of plantgrowth-promoting rhizobacteria (PGPR) that are capable of improving thegrowth of plants, and also have disease- and pest-controlling activity.From an analysis of genome sequences from the best-performing Bacillusamyloliquefaciens subspecies plantarum PGPR strains, the inventorsidentified some genetically encoded functions that are always presentwithin these Bacillus PGPR strains and are not present in other Bacillusspecies that are not plant-related. In particular, these PGPR strainscan use sugars derived from plant pectin as a carbon and/or energysource. By supplementing pectin on plant seeds that are inoculated withBacillus spores, or by supplementing the amount of pectin available forBacillus PGPR strain post-seed germination, this will result in anenhancement of 1) the Bacillus strain colonization of the plantrhizosphere and/or 2) better persistence of Bacillus within the plantrhizosphere and/or 3) better plant growth performance in response toPGPR strain+pectin administration and/or 4) better biological control ofdisease (e.g., bacteria, fungi, viruses) or pests (e.g., nematodes) as aresult of PGPR strain+pectin administration.

PGPR

The term “plant growth promoting rhizobacteria” or “PGPR” refers to agroup of bacteria that colonize plant roots, and in doing so, promoteplant growth and/or reduce disease or damage from predators. Bacteriathat are PGPR may belong to genera including, but not limited toActinobacter, Alcaligenes, Bacillus, Burkholderia, Buttiauxella,Enterobacter, Klebsiella, Kluyvera, Pseudomonas, Rahnella, Ralstonia,Rhizobium, Serratia, Stenotrophomonas, Paenibacillus, andLysinibacillus. The PGPR utilized in the disclosed methods andcomposition may be a single strain, species, or genus of bacteria or maycomprise a mixture of bacterial strains, species, or genera. Forexample, the PGPR may be selected from genera including, but not limitedto, Actinobacter, Alcaligenes, Bacillus, Burkholderia, Buttiauxella,Enterobacter, Klebsiella, Kluyvera, Pseudomonas, Rahnella, Ralstonia,Rhizobium, Serratia, Stenotrophomonas, Paenibacillus, andLysinibacillus.

The genus Bacillus as used herein refers to a genus of Gram-positive,rod-shaped bacteria which are members of the division Firmicutes. Understressful environmental conditions, the Bacillus bacteria produce ovalendospores that can stay dormant for extended periods. Bacillus bacteriamay be characterized and identified based on the nucleotide sequence oftheir 16S rRNA or a fragment thereof (e.g., approximately a 1000 nt,1100 nt, 1200 nt, 1300 nt, 1400 nt, or 1500 nt fragment of 16S rRNA orrDNA nucleotide sequence). Bacillus bacteria may include, but are notlimited to B. acidiceler, B. acidicola, B. acidiproducens, B. aeolius,B. aerius, B. aerophilus, B. agaradhaerens, B. aidingensis, B. akibai,B. alcalophilus, B. algicola, B. alkalinitrilicus, B. alkalisediminis,B. alkalitelluris, B. altitudinis, B. alveayuensis, B.amyloliquefaciens, B. anthracis, B. aquimaris, B. arsenicus, B.aryabhattai, B. asahii, B. atrophaeus, B. aurantiacus, B. azotoformans,B. badius, B. barbaricus, B. bataviensis, B. beijingensis, B.benzoevorans, B. beveridgei, B. bogoriensis, B. boroniphilus, B.butanolivorans, B. canaveralius, B. carboniphilus, B. cecembensis, B.cellulosilyticus, B. cereus, B. chagannorensis, B. chungangensis, B.cibi, B. circulans, B. clarkii, B. clausii, B. coagulans, B.coahuilensis, B. cohnii, B. decisifrondis, B. decolorationis, B.drentensis, B. farraginis, B. fastidiosus, B. firmus, B. flexus, B.foraminis, B. fordii, B. fortis, B. fumarioli, B. funiculus, B.galactosidilyticus, B. galliciensis, B. gelatini, B. gibsonii, B.ginsengi, B. ginsengihumi, B. graminis, B. halmapalus, B. halochares, B.halodurans, B. hemicellulosilyticus, B. herbertsteinensis, B. horikoshi,B. horneckiae, B. horti, B. humi, B. hwajinpoensis, B. idriensis, B.indicus, B. infantis, B. infernus, B. isabeliae, B. isronensis, B.jeotgali, B. koreensis, B. korlensis, B. kribbensis, B. krulwichiae, B.lehensis, B. lentus, B. licheniformis, B. litoralis, B. locisalis, B.luciferensis, B. luteolus, B. macauensis, B. macyae, B. mannanilyticus,B. marisflavi, B. marmarensis, B. massiliensis, B. megaterium, B.methanolicus, B. methylotrophicus, B. mojavensis, B. muralis, B.murimartini, B. mycoides, B. nanhaiensis, B. nanhaiisediminis, B.nealsonii, B. neizhouensis, B. niabensis, B. niacini, B. novalis, B.oceanisediminis, B. odysseyi, B. okhensis, B. okuhidensis, B. oleronius,B. oshimensis, B. panaciterrae, B. patagoniensis, B. persepolensis, B.plakortidis, B. pocheonensis, B. polygoni, B. pseudoalcaliphilus, B.pseudofirmus, B. pseudomycoides, B. psychrosaccharolyticus, B. pumilus,B. qingdaonensis, B. rigui, B. runs, B. safensis, B. salarius, B.saliphilus, B. schlegelii, B. selenatarsenatis, B. selenitireducens, B.seohaeanensis, B. shackletonii, B. siamensis, B. simplex, B. siralis, B.smithii, B. soli, B. solisalsi, B. sonorensis, B. sporothermodurans, B.stratosphericus, B. subterraneus, B. subtilis, B. taeansis, B.tequilensis, B. thermantarcticus, B. thermoamylovorans, B.thermocloacae, B. thermolactis, B. thioparans, B. thuringiensis, B.tripoxylicola, B. tusciae, B. vallismortis, B. vedderi, B. vietnamensis,B. vireti, B. wakoensis, B. weihenstephanensis, B. xiaoxiensis, andmixtures or blends thereof.

The PGPR and inoculants thereof disclosed herein may include B.amyloliquefaciens or a Bacillus species that is closely related to B.amyloliquefaciens. A Bacillus species that is closely related to B.amyloliquefaciens may be defined as a species having a 16S rDNA sequencecomprising SEQ ID NO:26 or comprising a 16S rDNA sequence having atleast about 98% or 99% sequence identity to SEQ ID NO:26. The PGPRpreferably is B. amyloliquefaciens subspecies plantarum or a Bacillusspecies that is closely related to B. amyloliquefaciens subspeciesplantarum. B. amyloliquefaciens subspecies plantarum is a subspecies ofB. amyloliquefaciens which is colonizes plant roots and typicallyexhibits amylase activity. Suitable PGPR strains for the disclosedmethods and compositions may include PGPR strains having a gyrB genethat exhibits sequence identity to the gyrB gene from strains ofBacillus amyloliquefaciens subspecies plantarum. In some embodiment, thePGPR strain utilized in the disclosed methods and compositions has atgyrB gene having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or100% sequence identity to the polynucleotide sequence of SEQ ID NO:25,which is the polynucleotide sequence of the gyrB gene from strains ofBacillus amyloliquefaciens subspecies plantarum.

Suitable strains of B. amyloliquefaciens subspecies plantarum for use inthe disclosed compositions and methods may include but are not limitedto Bacillus amyloliquefaciens subsp. plantarum AS43.3, Bacillusamyloliquefaciens subsp. plantarum TrigoCor1448, Bacillusamyloliquefaciens subsp. plantarum UCMB5033, Bacillus amyloliquefacienssubsp. plantarum UCMB5113, Bacillus amyloliquefaciens subsp. plantarumEBL11, Bacillus amyloliquefaciens subsp. plantarum W2, Bacillusamyloliquefaciens subsp. plantarum UCMB5036, Bacillus amyloliquefacienssubsp. plantarum IT-45, Bacillus amyloliquefaciens subsp. plantarumUASWS BA1, Bacillus amyloliquefaciens subsp. plantarum LFB 112, Bacillusamyloliquefaciens subsp. plantarum CAUB946, Bacillus amyloliquefacienssubsp. plantarum M27, Bacillus amyloliquefaciens subsp. plantarum B1895,Bacillus amyloliquefaciens subsp. plantarum SQR9, Bacillusamyloliquefaciens subsp. plantarum AH159-1, Bacillus amyloliquefacienssubsp. plantarum DC-12, Bacillus amyloliquefaciens subsp. plantarum YAUB9601-Y2, Bacillus amyloliquefaciens subsp. plantarum Y2, Bacillusamyloliquefaciens subsp. plantarum EGD_AQ14, Bacillus amyloliquefacienssubsp. plantarum NAU-B3, Bacillus amyloliquefaciens subsp. plantarumFZB42, Bacillus amyloliquefaciens subsp. plantarum CC 178, Bacillusamyloliquefaciens subsp. plantarum AP79, Bacillus amyloliquefacienssubsp. plantarum AP71, Bacillus amyloliquefaciens subsp. plantarumAP143, Bacillus amyloliquefaciens subsp. plantarum AP193, Bacillusamyloliquefaciens subsp. plantarum AB01, and Bacillus amyloliquefacienssubsp. plantarum GB03.

Suitable PGPR strains and inoculants thereof for the methods andcompositions disclosed herein may include PGPR strains that express oneor more proteins associated with pectin metabolism. In some embodiments,the PGPR strain may express one or more proteins associated with pectinmetabolism, which may include but are not limited to proteins encoded bya gene selected from the group consisting of uxaA (altronatedehydratase), uxaB (altronate oxidoreductase), uxaC (uronate isomerase),uxaA (mannonate dehydratase, uxuB (D-mannonate oxidoreductase), kdgA(4-hydroxy-2-oxoglutarate aldolase), kdgK(2-dehydro-3-deoxygluconokinase), exuR (hexuronate utilization operontranscriptional repressor), exuT (hexuronate transporter), andcombinations thereof. In some embodiments, the PGPR strain may expressone or more pectinase enzymes selected from a group consisting of pectinlyase, pectate lyase, polygalacturonase, and pectin esterase.

The uxaA gene encodes an enzyme which is an altronate dehydratase(EC:4.2.1.7) which converts D-altronate to 2-dehydro-3-deoxy-D-gluconateand water. Therefore, suitable PGPR strains and inoculants thereof forthe methods and composition disclosed herein may include a PGPR strainthat expresses altronate dehydratase. SEQ ID NO:1 provides thepolynucleotide sequence encoding for altronate dehydratase. SEQ ID NO:2provides the amino acid sequence for altronate dehydratase.

The uxaB gene encodes an enzyme which is an altronate oxidoreductase(EC:5.3.1.12) which converts D-altronate and NAD⁺ to D-tagaturonate andNADH. Therefore, suitable PGPR strains and inoculants thereof for themethods and composition disclosed herein may include a PGPR strain thatexpresses altronate oxidoreductase. SEQ ID NO:3 provides thepolynucleotide sequence encoding for altronate oxidoreductase. SEQ IDNO:4 provides the amino acid sequence for altronate oxidoreductase.

The uxaC gene encodes an enzyme which is an uronate isomerase(EC:1.3.1.12) which converts D-glucuronate to D-fructuronate and whichconverts D-galacturonate to D-tagaturonate. Therefore, suitable PGPRstrains and inoculants thereof for the methods and composition disclosedherein may include a PGPR strain that expresses uronate isomerase. SEQID NO:5 provides the polynucleotide sequence encoding for altronateoxidoreductase. SEQ ID NO:6 provides the amino acid sequence foraltronate oxidoreductase.

The uxuA gene encodes an enzyme which is a mannonate dehydratase(EC:4.2.1.8) which converts D-mannonate to2-dehydro-3-deoxy-D-gluconate. Therefore, suitable PGPR strains andinoculants thereof for the methods and composition disclosed herein mayinclude a PGPR strain that expresses mannonate dehydratase. SEQ ID NO:7provides the polynucleotide sequence encoding for mannonate dehydratase.SEQ ID NO:8 provides the amino acid sequence for mannonate dehydratase.

The uxuB gene encodes an enzyme which is a D-mannonate oxidoreductase(EC:1.1.1.57) which converts D-mannonate and NAD⁺ to D-fructuronate andNADH. Therefore, suitable PGPR strains and inoculants thereof for themethods and composition disclosed herein may include a PGPR strain thatexpresses D-mannonate oxidoreductase. SEQ ID NO:9 provides thepolynucleotide sequence encoding for altronate oxidoreductase. SEQ IDNO:10 provides the amino acid sequence for altronate oxidoreductase.

The kdgA gene encodes an enzyme which is a 4-hydroxy-2-oxoglutaratealdolase (EC 4.1.3.16) which converts 4-hydroxy-2-oxoglutarate topyruvate and glyoxylate, and which converts2-dehydro-3-deoxy-6-phosphate-D-gluconate to pyruvate andD-glyceraldehyde 3-phosphate. Therefore, suitable PGPR strains andinoculants thereof for the methods and composition disclosed herein mayinclude a PGPR strain that expresses 4-hydroxy-2-oxoglutarate aldolase.SEQ ID NO:11 provides the polynucleotide sequence encoding for4-hydroxy-2-oxoglutarate aldolase. SEQ ID NO:12 provides the amino acidsequence for 4-hydroxy-2-oxoglutarate aldolase.

The kdgK gene encodes an enzyme which is 2-dehydro-3-deoxygluconokinase(EC 2.7.1.45) which phosphorylates 2-keto-3-deoxygluconate (KDG) toproduce 2-keto-3-deoxy-6-phosphogluconate (KDPG). Therefore, suitablePGPR strains and inoculants thereof for the methods and compositiondisclosed herein may include a PGPR strain that expresses2-dehydro-3-deoxygluconokinase. SEQ ID NO:13 provides the polynucleotidesequence encoding for 2-dehydro-3-deoxygluconokinase. SEQ ID NO:14provides the amino acid sequence for 2-dehydro-3-deoxygluconokinase.

The exuR gene encodes a hexuronate utilization operon transcriptionalrepressor. Therefore, suitable PGPR strains and inoculants thereof forthe methods and composition disclosed herein may include a PGPR strainthat expresses a hexuronate utilization operon transcriptionalrepressor. SEQ ID NO:15 provides the polynucleotide sequence encodingfor a hexuronate utilization operon transcriptional repressor. SEQ IDNO:16 provides the amino acid sequence for a hexuronate utilizationoperon transcriptional repressor.

The exuT gene encodes a hexuronate transporter which exhibits hexuronatetransmembrane transporter activity. Therefore, suitable PGPR strains andinoculants thereof for the methods and composition disclosed herein mayinclude a PGPR strain that expresses a hexuronate transporter. SEQ IDNO:17 provides the polynucleotide sequence encoding for a hexuronatetransporter. SEQ ID NO:18 provides the amino acid sequence for ahexuronate transporter.

In some embodiments, the PGPR strain may express one or more pectinaseenzymes selected from a group consisting of pectin lyase (EC 4.2.2.10),pectate lyase (EC 4.2.2.2), polygalacturonase (EC 3.2.1.15), and pectinesterase (EC 3.1.1.11). SEQ ID NO:19 provides the polynucleotidesequence encoding for a pectate lyase precursor. SEQ ID NO:20 providesthe amino acid sequence for a pectate lyase precursor. SEQ ID NO:21provides the polynucleotide sequence encoding for a pectin-lyase likeprotein. SEQ ID NO:22 provides the amino acid sequence for apectin-lyase like protein. SEQ ID NO:23 provides the polynucleotidesequence encoding for a pectin lyase. SEQ ID NO:24 provides the aminoacid sequence for a pectin lyase.

“Percentage sequence identity” may be determined by aligning twosequences of equivalent length using the Basic Local Alignment SearchTool (BLAST) available at the National Center for BiotechnologyInformation (NCBI) website (i.e., “b12seq” as described in Tatiana A.Tatusova, Thomas L. Madden (1999), “Blast 2 sequences—a new tool forcomparing protein and nucleotide sequences”, FEMS Microbiol Lett.174:247-250, incorporated herein by reference in its entirety). Forexample, percentage sequence identity between SEQ ID NO:1 and anothersequence for comparison may be determined by aligning these twosequences using the online BLAST software provided at the NCBI website.

“Percentage sequence identity” between two deoxyribonucleotide sequencesmay also be determined using the Kimura 2-parameter distance model whichcorrects for multiple hits, taking into account transitional andtransversional substitution rates, while assuming that the fournucleotide frequencies are the same and that rates of substitution donot vary among sites (Nei and Kumar, 2000) as implemented in the MEGA 4(Tamura K, Dudley J, Nei M & Kumar S (2007) MEGA4: MolecularEvolutionary Genetics Analysis (MEGA) software version 4.0. MolecularBiology and Evolution 24:1596-1599), preferably version 4.0.2 or later.The gap opening and extension penalties are set to 15 and 6.66respectively. Terminal gaps are not penalized. The delay divergentsequences switch is set to 30. The transition weight score is 35 set to0.5, as a balance between a complete mismatch and a matched pair score.The DNA weight matrix used is the IUB scoring matrix where x′s and n′sare matches to any IUB ambiguity symbol, and all matches score 1.9, andall mismatched score O.

Pectin and Pectin-Related Saccharides

The disclosed compositions and methods include or utilize pectin orpectin-derived sugars in order to sugars to enhance the efficacy of PGPRin regard to promoting plant growth and plant health. “Pectin” is aheteropolysaccharide found natively in the primary cell walls ofterrestrial plants having a typical molecular weight of 60,000-130,000g/mol, which varies based on the origin of the pectin and the extractionconditions. As used herein, “pectin” is meant to include extractedpectin that has been extracted from its native condition (e.g.,extracted pectin from primary cell walls of terrestrial plants).

The compositions and methods disclosed herein may comprise and/orutilize a relatively high molecular weight polysaccharide such as arelatively high molecular weight pectin. In some embodiments, thecompositions and methods disclosed herein comprise and/or utilize pectinhaving an average molecular weight of at least about 1000, 2000, 5000,10000, 15000, 20000, 25000, 30000, 35000, 40000, 45000, 50000, 55000,60000, 65000, 70000, 75000, 80000, 85000, 90000, 95000, 100000, 105000,110000, 115000, 120000, 125000, 130000 g/mol or higher. In someembodiments, the relatively high molecular weight polysacchariderepresents at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, or higher of the total amount of carbohydrate in a composition ascontemplated herein.

The disclosed composition and methods may include and/or utilize pectingand/or pectin derived sugars in any form. In some embodiments, thepectin and/or pectin derivated sugars are in powder form. The powderform may be utilized to prepare a solution of the pectin and/or pectinderivated sugars. Solutions of pectin prepared for use in the presentlydisclosed methods may have a concentration (w/w) of about 0.0001%,0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, or5%, or solutions of pectin prepared for use in the presently disclosedmethods may have a concentation of pectin within a range bounded by anyof these percentage values (e.g., within a range of 0.01%-1%). Asolution thus prepared may be utilized in the methods disclosed herein,including methods for treating plants, increasing nodulation in legumes,and/or treating animals.

The pectin or pectin-related saccharides utilized in the disclosedcomposition and methods may be isolated or substantially purified. Theterms “isolated” or “substantially purified” refers to pectin orpectin-related saccharides that have been removed from a naturalenvironment and have been isolated or separated, and are at least 60%free, preferably at least 75% free, and more preferably at least 90%free, even more preferably at least 95% free, and most preferably atleast 100% free from other components with which they were naturallyassociated, which other components may include but are not limited tocellulose or other non-pectin polysaccharides.

Although the composition of pectin may vary among plants, pectintypically has a composition in which D-galacturonic acid is the mainmonomeric constituent (i.e., typically D-galacturonic acidrepresents >50% of the monomeric constituents of pectin). TheD-galacturonic residues of pectin optionally may be substituted withD-xylose or D-apiose to form xylogalacturonan and apiogalacturonan,respectively, branching from a D-galacturonic acid residue. So-called“rhamnogalcturonan pectins” contain a backbone of repeatingdisaccharides of D-galacturonic acid and L-rhamnose. Pectins andpectin-derived products suitable for use in the presently disclosedcompositions and methods may include pectin in which D-galacturonic acidrepresents >50% of the monomeric constituents of the pectin, optionallywhere one or more of the D-galacturonic residues of pectin aresubstituted with D-xylose or D-apiose to form xylogalacturonan andapiogalacturonan, respectively, branching from a D-galacturonic acidresidue. Pectins and pectin-derived products suitable for use in thepresently disclosed compositions and methods may include so-called“rhamnogalcturonan pectins” that contain a backbone of repeatingdisaccharides of D-galacturonic acid and L-rhamnose.

In nature, the majority of carboxyl groups of galacturonic acid inpectin are esterified with methanol (i.e., >50% and as much as 80% ofthe carboxyl groups of galacturonic acid in pectin are esterified withmethanol). During extraction, this percentage may decrease whereextraction may result in hydrolysis of the ester bond, and extractedpectins may be categorized as high-ester versus low-ester pectins having<50% of galacturonic acid residues being esterified. Non-esterifiedgalacturonic acid units can be either free acids (i.e., carboxyl groups)or salts with sodium, potassium, or calcium (i.e., galacturonate salts).Pectins and pectin-derived products suitable for use in the presentlydisclosed compositions and methods may include pectins in which themajority of carboxyl groups of galacturonic acid in pectin areesterified with methanol (i.e., >50% and as much as 80% of the carboxylgroups of galacturonic acid in pectin are esterified with methanol).After extraction, pectins and pectin-derived products suitable for usein the presently disclosed compositions and methods may includeextracted pectins (e.g., high-ester pectins or low-ester pectins having<50% of galacturonic acid residues being esterified).

In nature, D-galacturonic acid may be synthesized from D-gluconoric acidderivatives (e.g., from UDP-D-glucuronate via 4-epimerization) andconversely, D-galacturonic acid in pectin may be metabolized to formD-gluconoric acid derivatives (e.g., 5-dehydro-4-deoxy-D-glucuronate viaoligogalacturonate lysis). As used herein, pectin-related saccharidesinclude pectin-derived saccharides such as hydrolyzed pectin,D-galacturonic acid (or D-galacturonate salts), and D-gluconoric acid(or D-gluconorate salts), polymers thereof, or combinations thereof.

The compositions and methods disclosed herein may include or utilize asaccharide that is a substrate for an enzyme or transporter encoded by agene selected from the group consisting of uxaA (altronate dehydratase),uxaB (altronate oxidoreductase), uxaC (uronate isomerase), uxuA(mannonate dehydratase), uxuB (D-mannonate oxidoreductase), kdgA(4-hydroxy-2-oxoglutarate aldolase), kdgK(2-dehydro-3-deoxygluconokinase), exuR (hexuronate utilization operontranscriptional repressor), exuT (hexuronate transporter), andcombinations thereof. The compositions and methods disclosed herein mayinclude or utilize a saccharide that is a substrate for a pectinaseenzyme (e.g., a pectinase enzyme selected from a group consisting ofpectin lyase, pectate lyase, polygalacturonase, and pectin esterase).

Substrates as such may include but are not limited to saccharidesderived from pectin such as D-galacturonate and D-glucuronate. Thesaccharide may comprise a mixture of sugars or the saccharide maycomprise a heteropolysaccharide. In embodiments in which the saccharideis a heterogeneous mixture of sugars or the saccharide is aheteropolysaccharide, preferably D-galacturonate monomeric units,D-glucuronate monomeric units, or the sum of D-galacturonate monomericunits and D-glucuronate monomeric unitsrepresent >50%, >60%, >70%, >80%, >90%, or >95% of total monomeric unitsin the heterogeneous mixture of sugars or the heteropolysaccharide.

The disclosed pectin and pectin-related substances may include syntheticpectin. Synthetic pectin may include pectin synthesized by polymerizingpectin monomers (e.g., uronic acid) in vitro to form pectin-likesubstance referred to as synthetic pectin. (See, e.g., U.S. Pat. No.2,156,223. Furthermore, the disclosed pectin and pectin-relatedsubstances may include naturally and non-naturally occurring polyuronicacids.

In the disclosed methods and compositions, pectin may be present at adesirable concentration, for example, in soil surrounding a plant, in aseed coating, or in animal feed. When pectin is administered to soil,the pectin may be administered, for example, to achieve a concentrationin soil (w/w) of about 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%,0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5%, or to achieve a concentration in soilwithin a concentration range bounded by any of these percentage values(e.g., within a range of 0.001%-0.01%). When pectin is present in a seedcoating, the pectin may be present at a concentration (w/w) in thecoating of about 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%,20%, 30%, 40%, 50%, or higher, or the pectin may be present atconcentration (w/w) in the coating within a concentration range boundedby any two of these values (e.g., within a range of 0.1%-1%). Whenpectin is present in animal feed, the pectin may be present at aconcentration (w/w) in the animal feed of about 0.005%, 0.01%, 0.05%,0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10% or higher or the pectin may bepresent in the animal feed at within range concentration bounded by anytwo of these values (e.g., within a range of 0.1%-1%).

Inoculants

The presently disclosed PGPR may be formulated as an inoculant for aplant. The term “inoculant” means a preparation that includes anisolated culture of a PGPR and optionally a carrier. Inoculantscomprising PGPR and carriers are known in the art. (See, e.g., Bashan,“Inoculants of Plant Growth-Promoting Bacteria for use in Agriculture,”Biotechnology Advances, Vol. 16, No. 4, pp. 729-770, 1998). PGPRinoculants may be administered to plants (e.g. to the roots of plants),to seeds (e.g., as a coating for the seed or at the time that the seedis planted), or to soil (e.g., to soil surrounding plants to betreated).

A PGPR inoculant may be described as a formulation containing one ormore PRPR species in a carrier material, which may be an organiccarrier, an inorganic carrier, or a carrier synthesized from definedmolecules. Optionally, the carrier may be sterile or sterilized prior tobe formulated with the PGPR to form the PGPR inoculant. Preferably, thecarrier is nontoxic, biodegradable and nonpolluting. In the disclosedinoculants comprising a pectin saccharide, the pectin saccharideoptionally may function as a carrier or optionally the inoculants maycomprise a carrier other than the pectin saccharide.

The carrier of the PGPR inoculant is the delivery vehicle for the livePGPR to the plant, seeds, or soil. The carrier represent is the majorportion by volume or weight of the inoculant. Suitable carriers mayinclude liquids, powders (e.g., having an average effective particlediameter of 0.075 to 0.25 mm), granulars (e.g., having an averageeffective particle diameter of 0.35 to 1.18 mm), and slurries which havethe capacity to deliver a sufficient number of viable PGPR cells to theplant, seeds, or soil. Preferably, the carrier extends the shelf-life ofthe PGPR (e.g., such that the PGPR has a shelf-life of at least 1 or 2years at room temperature). Examples of carriers include but are notlimited to peat, coal, clays, inorganic soil material, plant wastematerials, composts, farmyard manure, soybean meal, soybean oil, peanutoil, wheat bran, inert materials such as vermiculite, perlite,phosphate, polyacrylamide, alginate beads, oil-dried bacteria. In someembodiments, the PGPR may be encapsulated by a carrier, for example,where the carrier is a carbohydrate that forms a matrix around the PGPR.

The PGPR utilized in the disclosed composition and methods may beisolated or substantially purified. The terms “isolated” or“substantially purified” refers to PGPR that have been removed from anatural environment and have been isolated or separated, and are atleast 60% free, preferably at least 75% free, and more preferably atleast 90% free, even more preferably at least 95% free, and mostpreferably at least 100% free from other components with which they werenaturally associated. An “isolated culture” refers to a culture of thePGPR that does not include significant amounts of other materials suchas other materials which normally are found in soil in which the PGPRgrows and/or from which the PGPR normally may be obtained. An “isolatedculture” may be a culture that does not include any other biological,microorganism, and/or bacterial species in quantities sufficient tointerfere with the replication of the “isolated culture.” Isolatedcultures of PGPR may be combined to prepare a mixed culture of PGPR.

The inoculant typically includes a suitable amount of PGPR relative tocarrier. In some embodiments, the inoculant includes 10²-10¹² cfu PGPRper ml carrier (or per gram carrier), or 10⁴-10¹⁰ cfu PGPR per mlcarrier (or per gram carrier), or 10⁶-10⁸ cfu PGPR per ml carrier (orper gram carrier). The composition may include additional additivesincluding buffering agents, surfactants, adjuvants, or coating agents.Suitable carriers may include, but are not limited to, water or otheraqueous solutions, slurries, solids (e.g., peat, wheat, bran,vermiculite, and pasteurized soil) or dry powders.

In the disclosed methods and compositions, PGPR may be present at adesirable concentration, for example, in soil surrounding a plant, in aseed coating, or in animal feed. In some embodiments where PGPR isapplied to soil, PGPR may be applied as a seedling root-dip or as a soildrench at a concentration of about 10²-10¹² cfu/ml, 10⁴-10¹⁰ cfu/ml, orabout 10⁶-10⁸ cfu/ml. In some embodiments where PGPR is present as acoating on a seed, suitable application concentrations may be between10²-10⁸ cfu per seed, preferably 10⁴-10⁷ cfu per seed. In someembodiments where PGPR is present in animal feed, the PGPR may bepresented at a concentration of at least about 10⁴ CFU/g of feed. Morepreferably, the spore-forming strain of the genus Bacillus is present inthe composition at a concentration of at least about 10⁵ CFU/g of feed.Even more preferably, the spore-forming strain of the genus Bacillus ispresent in the composition at a concentration of at least about 10⁶CFU/g of feed or per ml of water or at least about 10⁷ CFU/g of feed orper ml of water. A suitable concentration range may include 10⁴-10⁷CFU/g of feed or per ml of water or sub-ranges there within.

The disclosed inoculants and compositions may include additional agentsfor promoting plant growth and health, including additional agents forpromoting nodulation. The additional agents may include additionalbacterial inoculants, including, but not limited to, additionalrhizobacteria such as a nitrogen-fixing bacteria and/or aphosphate-solubilizing bacteria. Additional agents may include fungalinoculants, for example mycorrhizae. Additional agents may include plantnutrients such as nitrogen salts and/or phosphate salts and/or potassiumsalts.

Methods of Treating Plants, Seeds, or Soil

Also disclosed are methods of using pectin or pectin-related saccharidesto improve the efficacy of PGPR in regard to promoting growth or healthin plant. The disclosed methods for promoting growth or health in plantmay include, but are not limited to, methods of increasing nodulation inlegumes. The disclosed methods may include administering theabove-described inoculants comprising a PGPR and a pectin saccharide toplants, seeds, or soil. In some embodiments, the disclosed methods forimproving plant growth or plant health may include: (a) treating plants,seeds, or soil with a plant growth promoting rhizobacteria (PGPR) thatexpresses a protein associated with pectin metabolism and (b) treatingthe plants, seeds, or soil with a saccharide comprising pectin or apectin-related saccharide (e.g., hydrolyzed pectin, D-galacturonate,D-glucuronate, or mixtures thereof), where the plants, seeds, or soilmay be treated with the PGPR and the saccharide concurrently or ineither order (i.e., the PGPR may be administered before, concurrentlywith, or after the saccharide is administered). The PGPR and pectinsaccharide may be formulated as an inoculant and administeredconcurrently to treat plants (e.g., administered to the roots ofplants), to seeds (e.g., as a coating for seeds), or to soil (e.g., as asoil amendment).

The disclosed methods may be utilized to improve plant growth or planthealth by controlling soil-borne pests. Soil-borne pests controlled bythe disclosed methods may include but are not limited to nematodes andherbivorous insects. The disclosed methods may be utilized to improveplant growth or plant health by controlling or treating a disease.Disease controlled or treated by the disclosed methods may include butare not limited to a bacterial disease, a fungal disease, and a viraldisease.

The presently disclosed PGPR and pectin saccharide may be administeredas an inoculant for treating plants. The methods of treatmentcontemplated herein may include treating a plant directly includingtreating leaves, stems, or roots of the plant directly. The methods oftreatment contemplated herein may include treating seeds of the plant,e.g., coating the seeds prior to the seeds being planted to produce atreated plant. The methods contemplated herein also may include treatinga plant indirectly, for example, by treating soil or the environmentsurrounding the plant (e.g., in-furrow granular or liquid applications).Suitable methods of treatment may include applying an inoculantincluding the PGPR and the saccharide via high or low pressure spraying,drenching, and/or injection. Plant seeds may be treated by applying lowor high pressure spraying, coating, immersion, and/or injection. Afterplant seeds have been thusly treated, the seeds may be planted andcultivated to produce plants. Plants propagated from such seeds may befurther treated with one or more applications. Suitable applicationconcentrations may be determined empirically. In some embodiments wherethe PGPR and pectin saccharide are applied as a spray to plants,suitable application concentrations may include spraying 10⁶-10¹⁸ colonyforming units (cfu) per hectare of plants, more commonly 10⁷-10¹⁵ cfuper hectare. For coated seeds, in some embodiments, suitable applicationconcentrations may be between 10²-10⁸ cfu per seed, preferably 10⁴-10⁷cfu per seed. In other embodiments, the PGPR and pectin saccharide maybe applied as a seedling root-dip or as a soil drench at a concentrationof about 10²-10¹² cfu/ml, 10⁴-10¹⁰ cfu/ml, or about 10⁶-10⁸ cfu/ml.

Methods of Treating Animals

Bacillus species isolates cultured from plant rhizospheres have theability to utilize complex plant polysaccharides as a carbon and energysource. Increasingly, animal feeds are plant based and many of theplant-derived polysaccharides and other compounds (e.g. phytic acid) arenot readily degraded or utilized by fish, poultry or livestock. In fact,in many cases these plant-derived compounds such as phytic acid serve asan anti-nutrient that can make animals anemic. Using Bacillus or otherspecies that can degrade complex plant polysaccharides can promote feedconversion efficiency and these rhizosphere isolates are ideally suitedto help improve animal feeds and degrade phytic acid to improve animalnutrition.

As such, also disclosed are methods of using pectin or pectin-relatedsaccharides to improve the efficacy of PGPR in regard to promotinggrowth or health in animals. The disclosed methods may includeadministering the afore-described inoculants comprising a PGPR and apectin saccharide to animals (e.g., in the form of an animal feedcomposition such as a pelleted feed composition comprising theafore-described inoculants). In some embodiments, the disclosed methodsfor improving animal growth or animal health may include: (a)administering to an animal a plant growth promoting rhizobacteria (PGPR)that expresses a protein associated with pectin metabolism and (b)administering to the animal a pectin saccharide comprising pectin or apectin-related saccharides (e.g., hydrolyzed pectin, D-galacturonate,D-glucuronate, or mixtures thereof), where the animals may beadministered the PGPR and the pectin saccharide concurrently or ineither order (i.e., the PGPR may be administered before, concurrentlywith, or after the saccharide is administered).

Feed compositions comprising the PGPR and pectin saccharide may beadministered to animals orally. Oral administration includes, but is notlimited to, delivery in feed, water, by oral gavage or aerosol spray. Ifsupplied in an animal feed, the feed may comprise between 10⁴ and 10⁹cfu PGPR/gm of finished feed. Suitably the feed comprises between 10⁵and 5×10⁷ cfu PGPR/gm feed. The PGPR and pectin saccharide may be addedto the feed during production, after production by the supplier, or bythe person feeding the animals, just prior to providing the food to theanimals.

An animal feed composition may be prepared by forming a mixture of theanimal feed and an inoculant as discussed above, and then optionallyforming a compressed or pelleted animal feed from the mixture. Animalfeed suitable for preparing animal feed compositions as disclosed hereinmay include animal feed comprising plant material (e.g., hay straw,silage, grains (e.g., maize, soybean, wheat, oats, barley, sorghum, andrice), and legumes). Animal feed suitable for preraing animal feedcompositions as disclosed herein amy include fish products (e.g., fishoils and fish proteins).

The disclosed methods for promoting growth or health in animals may bepracticed in order to increase overall gastrointestinal health, improveproduction performance, and reduce enteric bacterial pathogens ofimportance to both animal health and human food safety. These PGPR andpectin saccharide may be added to animal diets at the rate of about 10⁴to 10⁹ PGPR per gram of finished feed for optimal inclusion rate, if thebacteria or probiotic compositions being administered continuously, anda higher inclusion rate may be necessary if the PGPR or the compositionsare provided intermittently. While administration though the feed is apreferred route of administration, the PGPR and pectin saccharide mayalso be administered via drinking water, through course spray, throughaerosol spray, or through any other means by which the agriculturalanimals may ingest these PGPR and pectin saccharide.

The disclosed methods may include methods of promoting growth or healthin aquatic animals, which may include farmed fish (e.g. catfish ortilapia) and crustaceans (e.g., shrimp). The term “catfish” refers to afish belonging to the genus Ictaluri. Catfish may include the speciesIctaluri punctatus Rafinesque.

Methods for Preparing the Disclosed Compositions and Inoculants

Also disclosed are methods of using pectin or pectin-related saccharidesto prepare compositions and inoculants as disclosed herein. The methodsmay include combining PGPR and pectin, which has been extracted frompectin-containing plant material, or pectin-related saccharides toprepare the disclosed compositions and inoculants. Optionally, a carriermay be combined with the PGPR and pectin or pectin-related saccharidesto prepare the disclosed compositions and inoculants.

In some embodiments, the methods may include combining 10²-10¹² cfu PGPRper ml carrier (or per gram carrier), or 10⁴-10¹⁰ cfu PGPR per mlcarrier (or per gram carrier), or 10⁶-10⁸ cfu PGPR per ml carrier (orper gram carrier). In some embodiments, the methods may includecombining pectin, which has been extracted from pectin-containing plantmaterial, or pectin-related saccharides may be combined with PGPR andoptionally a carrier to prepare the disclosed compositions andinoculants, wherein the pectin or pectin-related saccharides are presentin the prepared compositions and inoculants at a concentration of atleast about 0.1%, 0.5%, 1.0%, 1.5%, or 2.0% (w/w or w/v) to about 0.5%,1.0%, 1.5%, 2.0%, or 5.0% (w/w or w/v). In some embodiments, the methodsmay include combining PGPR and pectin at a concentration of about atleast about 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰ , 10¹¹, 10¹²,10¹³, or 10¹⁴ cfu PGPR per gram pectin or pectin-related saccharides, toabout 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴,10¹⁵ cfu PGPR per gram pectin or pectin-related saccharides (e.g.,ranges such as 10⁷ to 10¹² cfu PGPR per gram pectin or pectin-relatedsaccharides are contemplated herein). In the methods, additionaladditives including buffering agents, surfactants, adjuvants, andcoating agents may be combined with the PGPR, pectin or pectin-relatedsaccharides, and optional carrier in order to prepare the disclosedcompositions and inoculants. Compositions and inoculants prepared by theafore-disclosed methods also are contemplated herein.

EXAMPLES

The following Examples are illustrative and are not intended to limitthe scope of the claimed subject matter.

Example 1—Deciphering the Conserved Genetic Loci Implicated in PlantDisease through Comparative Genomics of Bacillus amyloliquefacienssubsp. plantarum Strains

Reference is made to Hossain et al., “Deciphering the conserved geneticloci implicated in plant disease through comparative genomics ofBacillus amyloliquefaciens subsp. plantarum strains,” Frontiers in PlantScience, 2015 Aug. 17; 6:631 doi: 10.3389/fpls.2015.00631. eCollection2015, (hereinafter referred to as “Hossain et al., Frontiers PlantScience 2015), the content of which is incorporated herein by referencein its entirety.

Abstract

To understand the growth-promoting and disease-inhibiting activities ofplant growth-promoting rhizobacteria (PGPR) strains, the genomes of 12Bacillus subtilis group strains with PGPR activity were sequenced andanalyzed. These B. subtilis strains exhibited high genomic diversity,whereas the genomes of B. amyloliquefaciens strains (a member of the B.subtilis group) are highly conserved. A pairwise BLASTp matrix revealedthat gene family similarity among Bacillus genomes ranges from 32-90%,with 2,839 genes within the core genome of B. amyloliquefaciens subsp.plantarum. Comparative genomic analyses of B. amyloliquefaciens strainsidentified genes that are linked with biological control andcolonization of roots and/or leaves, including 73 genes uniquelyassociated with subsp. plantarum strains that have predicted functionsrelated to signaling, transportation, secondary metabolite production,and carbon source utilization. Although B. amyloliquefaciens subsp.plantarum strains contain gene clusters that encode many differentsecondary metabolites, only polyketide biosynthetic clusters that encodedifficidin and macrolactin are conserved within this subspecies. Toevaluate their role in plant pathogen biocontrol, genes involved insecondary metabolite biosynthesis were deleted in B. amyloliquefacienssubsp. plantarum strain, revealing that difficidin expression iscritical in reducing the severity of disease, caused by Xanthomonasaxonopodis pv. vesicatoria in tomato plants. This Example definesgenomic features of PGPR strains and links them with biocontrol activityand with host colonization.

Introduction

Bacteria associated with plant roots that exert beneficial effects onplant growth and development are referred to as plant growth-promotingrhizobacteria (PGPR) (Kloepper and Schroth, 1978; Kloepper et al.,2004). Bacillus and Pseudomonas spp. are predominant among the diversebacterial genera that have been linked with PGPR activity (Podile andKishore, 2006). Members of the B. subtilis group, including B. subtilis,B. licheniformis, B. pumilus, B. amyloliquefaciens, B. atrophaeus, B.mojavensis, B. vallismortis, B. sonorensis, and B. tequilensis have beenidentified as PGPR strains for their capacity to stimulate plant growthand suppress pathogens within rhizosphere and phyllosphere (Kloepper etal., 2004; Hao et al., 2012; Kim et al., 2012). Strains of B.amyloliquefaciens are widely used for their positive effects on plantgrowth (Idriss et al., 2002). Reva et al. (Reva et al., 2004) reportedthat seven Bacillus isolates from plants or soil are closely related yetdistinct from B. amyloliquefaciens type strain DSM7^(T). In addition,these strains are more proficient for rhizosphere colonization thanother members of the B. subtilis group. GB03 (Nakkeeran et al., 2005),INR7 (Kokalis-Burelle et al., 2002) and FZB42 (Chen et al., 2007a) arePGPR strains within the Bacillus subtilis group that have been widelyused in different commercial formulations to promote plant growth.

In addition to promoting plant growth, PGPR strains may exhibitbiological control of plant diseases. Antibiosis, through the productionof inhibitory bioactive compounds, and induced systemic resistance arewidely reported biological control mechanisms of Bacillus spp. PGPRstrains (Ryu et al., 2004). PGPR Bacillus spp. strains produce diverseantimicrobial compounds including antibiotics (Emmert et al., 2004),volatile organic compounds (VOCs) (Yuan et al., 2012), and lipopeptides(Ongena et al., 2007) that are associated with the observed biocontrolactivity against plant pathogens. For example, B. amyloliquefaciensNJN-6 produces 11 VOCs that provide antifungal activity against Fusariumoxysporum f. sp. cubense (Yuan et al., 2012). Similarly, B. subtilsstrains produce lipopeptides (e.g. surfactin and fengycin), that inducesystemic resistance in bean plants (Ongena et al., 2007). PGPR strainsusually need to colonize plant roots extensively to exert plant growthpromoting effects using both direct and indirect mechanisms (Lugtenbergand Kamilova, 2009), extensive root colonization is not required forinduced systemic resistance (ISR) (Kamilova et al., 2005). In some PGPRstrains, root colonization is a prerequisite for biocontrol activitythrough antibiosis (Chin et al., 2000). For example, B.amyloliquefaciens subsp. plantarum FZB42 exerts growth promotingactivities through efficient colonization of plant roots (Fan et al.,2011). Previously, it has been demonstrated that over-expression ofgenes involved in phosphorylation of DegU, a two-component responseregulator of B. amyloliquefaciens strain SQR9, positively influencesroot colonization as well as other growth-promoting activities by PGPRstrains for controlling cucumber wilt disease (Xu et al., 2014).Moreover, the root colonization capacity of a poor root colonizer can beimproved by cloning genes that are required for efficient rootcolonization (Dekkers et al., 2000). Competitive root colonization byPGPR are controlled by many genes and/or genetic cluster(s) (Dietel etal., 2013), so identification of these genetic loci involved incompetitive root colonization are challenging if genome sequences arelacking for those PGPR strains (Lugtenberg and Kamilova, 2009). Analysisof additional PGPR strains will help elucidate the mechanisms ofcompetitive root colonization, antibiosis and ISR of PGPR strains andform a foundation for genetic engineering and other strategies toincrease the plant-growth promoting capacity of these bacteria.

In this Example, we sequenced the genomes of 12 Bacillus subtilis groupisolates from diverse locales. Comparative genomic analyses of PGPRstrains and control strains of the B. subtilis group without anyreported biocontrol activity against plant pathogens provides insightinto genomic features involved in PGPR activity. PGPR strain AP193,which inhibits growth of plant and animal bacterial pathogens (Ran etal., 2012), is an ideal candidate to evaluate the relative contributionof genes that are predicted to be involved in the biosynthesis ofbioactive secondary metabolites that could contribute to biocontrolactivity, specifically difficidin (dfnD mutant), surfactin (srfAAmutant), as well as all polyketides and lipopeptides produced bynon-ribosomal peptide synthesis, including difficidin (sfp mutant).Mutants were then tested for their ability to inhibit plant pathogens invitro and control bacterial spot disease in tomato.

Materials and Methods

Bacterial strains, plasmids and growth conditions. Bacterial strains andplasmids used in this Example are listed in Table 1. E. coli andBacillus strains were grown in Luria-Bertani (LB) medium; however, forelectrocompetent cell preparation, Bacillus amyloliquefaciens subsp.plantarum AP193 was grown in NCM medium (17.4 g K₂HPO₄, 11.6 g NaCl, 5 gglucose, 5 g tryptone, 1 g yeast extract, 0.3 g trisodium citrate, 0.05g MgSO₄.7H₂O and 91.1 g sorbitol in 1 L deionized water, pH 7.2). Forproduction of secondary metabolites, Bacillus cultures were grown for 48h at 30° C. in Tryptic Soy broth (TSB). In addition, ampicillin (100μg/ml), chloramphenicol (12.5 μg/ml) or erythromycin (200 μg/ml for E.coli or 5 μg/ml for Bacillus) were used as selective agents in growthmedia as required.

Sequencing, assembly and annotation. Next-generation sequencing ofBacillus spp. genomes was performed using Illumina and Roche 454sequencing platforms. Indexed Illumina libraries were prepared forstrains AP71, AP79, and ABO1 using Nextera DNA Sample Prep Kit(Epicentre, Madison, Wis.) and sequences were generated using anIllumina MiSeq with a 2×250 paired end sequencing kit. Barcoded Illuminalibraries for strains AP143, AP193, and AP254 were constructed using aNxSeq® DNA Sample Prep Kit (Lucigen, Middleton, Wis.) and sequenced atEnGenCore (Univ. of South Carolina) using the 454-pyrosequencingplatform. Genomic DNA library construction and sequencing for Bacillussubtilis GB03, Bacillus pumilus INR7, B. mojavensis KCTC 3706T, B.tequilensis KCTC 13622T, Bacillus siamensis KCTC 13613T, and B.sonorensis KCTC 13918T were conducted at the National Instrument Centerfor Environmental Management (Seoul, Republic of Korea), using theIllumina HiSeq 2000 sequencing platform. Sequence reads were trimmed forquality then assembled de novo using the CLC Genomics Workbench (CLCBio,Cambridge, Mass.). Gene prediction and annotation were performed usingGeneMark (Lukashin and Borodovsky, 1998) and the RAST annotation server(Aziz et al., 2008), respectively. The identity of individual openreading frames (ORFs) from secondary metabolite biosynthesis geneclusters was confirmed by BLASTx against the GenBank database. Genomesequence reads for strains AB01, AP71, AP79, AP143, AP193, AP254, GB03(Choi et al., 2014), INR7 (Jeong et al., 2014), KCTC 3706T, KCTC 13613T(Jeong et al., 2012), KCTC 13918T, and KCTC 13622T were deposited intothe Short Read Archive (SRA) at NCBI under the accession numbersSRR1176001, SRR1176002, SRR1176003, SRR1176004, SRR1176085, andSRR1176086, SRR1034787, SRR1141652, SRR1141654, SRR1144835, SRR1144836,and SRR1144837, respectively.

Determination of average nucleotide identity. Average nucleotideidentities (ANI) between genomes were calculated using an ANI calculatorthat estimates ANI according to the methods described previously (Goriset al., 2007).

Phylogenetic analysis of Bacillus species. For phylogenetic analysis,the gyrB gene sequence for each strain was retrieved from sequence data.(See Hossain et al., Frontiers Plant Science 2015, FIG. 1). StrainsAS43.3, FZB42, YAU B9601-Y2, CAU B946, and 5B6 were used asrepresentative strains of B. amyloliquefaciens subsp. plantarum; strainsDSM7, LL3 and TA208 were used as representative strains of B.amyloliquefaciens subsp. amyloliquefaciens. The gyrB phylogenetic treewas inferred with MEGA5.05 (Tamura et al., 2011) using Neighbor-Joining(Saitou and Nei, 1987) and Maximum Likelihood (ML) methods (Felsenstein,1981). All positions that contained gaps or missing data were eliminatedfrom the final dataset, resulting in 1911 bp positions of gyrB sequence.We used 729,383 bp of DNA to represent the conserved core genome foundacross 25 strains of the B. subtilis group, to generate a phylogenomictree using RAxML (v 7.2.7) (Pfeiffer and Stamatakis, 2010). Thephylogenomic tree was then visualized with iTOL (http://itol.embl.de)(Letunic and Bork, 2011).

BLAST matrix. The BLAST matrix algorithm was used for pairwisecomparison of Bacillus PGPR strain proteomes, using methods describedpreviously (Friis et al., 2010). The BLAST matrix determines the averagepercent similarity between proteomes by measuring the ratio of conservedgene families shared between strains to the total number of genefamilies within each strain. The absolute number of shared and combinedgene families for each strain was displayed in matrix output. Thismatrix shows the number of proteins shared between each proteome.

Core-genome analysis. The core-genome of 13 Bacillus spp. strains wasgenerated using coding and non-coding sequences. Whole genome sequencesfrom these strains were aligned using progressive Mauve (Darling et al.,2004), which identifies and aligns locally collinear blocks (LCBs) inthe XMFA format. LCBs from alignments were collected usingstripSubsetLCB s (http://gel.ahab.wise.edu/mauve/snapshots/), usingminimum lengths of 500bp. All LCBs were concatenated and converted tomultifasta format using a perl script. The same protocol was used toobtain all core sequences, with the exception that the minimum lengthsof LCBs were 50 bp, instead of 500 bp. The Bacillus spp. core genome wasobtained from the comparative alignment of all complete Bacillus spp.genomes available in the GenBank as of August 2014 (n=81 genomes). Thecore genome of the B. subtilis group was obtained from comparativeanalysis of 53 whole genomes of B. subtilis strains that included 41genomes obtained from GenBank and 12 PGPR genomes sequenced in thisExample. B. amyloliquefaciens species-level and B. amyloliquefacienssubsp. plantarum-level core genomes were generated from 32 B.amyloliquefaciens and 28 subsp. plantarum genomes. Core genomes wereexported to the CLC Genomics Workbench (v 4.9) for evaluation ofalignments and annotation using the RAST server (Aziz et al., 2008). Thelist of Bacillus spp. strains used for core genome determination isprovided in Table ?. Additionally, to identify GPR-specific core genes,raw sequence reads of PGPR strains sequenced in this Example weresequentially reference mapped against the genome sequence of non-PGPRstrain B. subtilis subsp. subtilis str. 168 according to methodsdescribed previously (Hossain et al., 2013).

Identification of core genes uniquely present in B. amyloliquefacienssubsp. plantarum strains. The aligned genome sequences of 32 B.amyloliquefaciens strains and 28 B. amyloliquefaciens subsp. plantarumstrains (which were included within the B. amyloliquefaciens strains)were analyzed using CLC Genomics Workbench to obtain the respectivespecies- and subsp.-level core genomes. Trimmed sequence reads of subsp.plantarum strain AP193 were reference mapped against the subsp.plantarum core genome to obtain core genome-specific sequence reads. Theparameters of reference mapping were as follows: mismatch cost=2,insertion cost=3, deletion cost=3, length fraction=0.5, andsimilarity=0.8. Sequence reads mapped to the subsp. plantarum coregenome were then mapped against the species amyloliquefaciens coregenome to obtain unmapped sequence reads. These unmapped sequence reads,represent the subsp. plantarum core genome that is absent in theamyloliquefaciens species-level core genome, were assembled de novousing CLC Genomics Workbench then the resulting contigs were uploaded toRAST for gene prediction and annotation. Each ORF, exclusively encodedby the plantarum core genome, was further confirmed for uniqueness usingBLASTn analysis against the genome sequences of 28 B. amyloliquefacienssubsp. plantarum and four B. amyloliquefaciens subsp. amyloliquefaciensstrains listed in Supplementary Table 1.

Prediction of secondary metabolite biosynthesis gene clusters in PGPRstrain AP193. Secondary metabolite biosynthesis gene clusters for strainAP193 were predicted using the secondary metabolite identification toolantiSMASH (Blin et al., 2013). Primer-walking PCR was used to fill gapsbetween contigs containing gene clusters encoding secondary metabolitebiosynthesis. Gene prediction and annotation were carried out byGeneMark (Lukashin and Borodovsky, 1998) and BLASTx (NCBI),respectively.

DNA manipulation and plasmid construction for PGPR strain AP193mutagenesis. Chromosomal DNA was isolated with the E.Z.N.A. BacterialDNA Isolation Kit (Omega Biotek, Atlanta, Ga.) and plasmids wereisolated with the E.Z.N.A. Plasmids Mini Kit II (Omega Biotek). Primersused in this Example are listed in Table 2. Gene deletion constructswere assembled using splicing through overlap extension PCR (Horton etal., 1989). The assembled products were gel purified with Gel/PCR DNAFragments Extraction Kit (IBI), digested with appropriate restrictionenzymes, and cloned into a pNZT1 vector to construct the deliveryplasmids for gene replacement.

In vitro plasmid methylation using cell free extract of Bacillusamyloliquefaciens subsp. plantarum AP193. To methylate plasmids prior totransformation into B. amyloliquefaciens subsp. plantarum AP193, themethod developed for Lactobacillus plantarum was used with minormodifications (Alegre et al., 2004). Cells from a 100 ml overnightculture of strain AP193 (OD₆₀₀=1.3-1.5) were pelleted by centrifugation(8000 xg), washed with 100 ml of chilled PENP buffer (10 mM potassiumphosphate, 10 mM EDTA, 50 mM NaCl and 0.2 mM PMSF, pH 7.0), and thenre-suspended to a final volume of 4 ml. Cells were disrupted byperforming two bursts (amplitude 50, pulse 3 and watts 25-30) for 5 mineach with a pause of 2 min, using a Vibra-Cell sonicator, and cooledwith ice to prevent overheating. Cell debris was removed bycentrifugation (8000 x g) at 4° C. and the extract was collected throughdecanting. Three ml aliquots of extract were mixed with 3 ml of glycerol(100% v/v) and 0.6 ml of bovine serum albumin (1 mg/ml), then stored at−20° C.

The DNA modification assay was performed in a final volume of 100 μl ofthe following: 53 μl TNE buffer [50 mM Tris (pH 7.5), 50 mM NaCl, 10 mMEDTA], 10 μl S-adenosylmethionine (0.8 mM), 2 μl BSA (5 mg/ml), 25 μlcell free extract derived from strain AP193 and 10 μl plasmid DNAextracted from E. coli K12 ER2925 (0.5-1 μg/μl). The mixture wasincubated at 37° C. for 16 h. Methylated DNA was extracted with a DNAClean & Concentrator Kit (Zymo Research, Calif.), then re-suspended inwater and stored at −20° C.

Electrotransformation of B. amyloliquefaciens subsp. plantarum AP193.For preparation of electrocompetent cells, strain AP193 was grownovernight in TSB, then diluted 100-fold in NCM to inoculate asubculture. The culture was grown at 37° C. on a rotary shaker until theOD₆₀₀ reached 0.7. The cell culture was cooled on ice for 15 min andsubjected to centrifugation at 8000×g for 5 min at 4° C. After washingfour times with ice cold ETM buffer (0.5 M sorbitol, 0.5 M mannitol, and10% glycerol), electrocompetent cells were re-suspended in 1/100 volumeof the original culture (Zhang et al., 2011). For electroporation, 100μl of cells were mixed with 100 ng of plasmid DNA in an ice-coldelectroporation cuvette (1 mm electrode gap). Cells were exposed to asingle 21 kV/cm pulse generated by Gene-Pulser (Bio-Rad Laboratories)with the resistance and capacitance set as 200Ω and 3 μF, respectively.The cells were immediately diluted into 1 ml of recovery medium (NCMplus 0.38M mannitol) (Zhang et al., 2011) and shaken gently at 30° C. or37° C. for 3 h to allow expression of the antibiotic resistance genes.Aliquots of the recovery culture were then spread onto LB agarsupplemented with appropriate antibiotics.

Two-step replacement recombination procedure for the modification of thestrain AP193 genome. A two-step replacement recombinationwas performedas previously described, with minor modifications (Zakataeva et al.,2010). To integrate the plasmid into AP193′s chromosome, a singlecrossover between the target gene and the homologous sequence on theplasmid must occur. To do this, AP193 that contained a delivery plasmidwith thedeletion construct was first grown in LB broth for 24 h at 37°C. (a non-permissive temperature for plasmid replication). Next, theculture was serially diluted, plated onto LB agar plates witherythromycin, and incubated at 37° C. Clones were screened by colony PCRusing two sets of primers. Each set of primers anneals sequencesspecific to one of the homologous fragments and to the chromosomalregion just outside of the other homologous fragment. If PCR productshad a reduced size, relative to the wild-type genotype for either primerset, this indicated successful chromosomal integration of the plasmid.In the second step, clones of the integrant were cultured with aerationin LB at 30° C. for 24-48 h to initiate the second single-crossoverevent, resulting in excision of the plasmid, yielding erythromycinsensitive (EmS) clones with either a parental or a mutant allele on thechromosome. Colony PCR was used to examine the presence of desiredmutations by primer sets that flank the deleted sequence.

Construction of strain AP193 mutants defective in secondary metabolitebiosynthesis. All mutant strains generated in this Example are indicatedin Table 1. The disruption of the dfnD gene was achieved as follows: DNAfragments corresponding to positions −867 to +247 and +643 to +1570 withrespect to the dfnD translation initiation site were PCR amplified usingAP193 genomic DNA as a template. The two fragments were then assembledby fusion PCR. A frameshift mutation was introduced during fusion toensure complete disruption of the gene. The deletion construct wasdigested with Xhol and Spel, then cloned into pNZT1, yielding pNZ-dif.The plasmid was methylated in vitro as described above and introducedinto strain AP193 by electroporation. Once introduced into strain AP193,plasmid pNZ-dif generated the isogenic mutant AP193ΔdfnD by two-stepreplacement recombination.

To generate the sfp deletion mutant, DNA fragments corresponding topositions −781 to +29, with respect to the sfp translation initiationsite, and +95 to +935, with respect to the sfp translation terminationsite, were PCR amplified using AP193 genomic DNA as template, assembledby fusion PCR, digested with HindIII and Pstl, and cloned into pNZT1 toconstruct pNZ-sfp. The plasmid pNZ-sfp was used to generate mutantAP193Δsfp using procedures described above.

The ΔsrfAA mutant was obtained as follows: DNA fragments correspondingto positions +5375 to +6091 and +6627 to +7366, with respect to thesrfAA translation initiation site, were PCR-amplified, fused by fusionPCR, digested with HindIII and Pstl and cloned into pNZT1 as pNZ-srf.Similarly, a frameshift mutation was introduced during the fusion of theupstream and downstream fragments of the target deletion sequence toensure complete disruption of the gene. The plasmid pNZ-srf was used togenerate mutant AP193ΔsrfAA using procedures described above.

In vitro antimicrobial activities of PGPR strain AP193 and its mutantsagainst plant pathogens. Plant pathogens Pseudomonas syringe pv. tabaci,Rhizobium radiobacter, Xanthomonas axonopodis pv. vesicatoria, andXanthomonas axonopodis pv. campestris were grown in TSB until the OD₆₀₀reached 1.0. The wild type strain AP193, as well as the three isogenicmutants ΔdfnD, Δsfp, and ΔsrfAA developed in this Example, were grown at30° C. in TSB for 48 h at 220 rpm. Cultures were then centrifuged at10,000×g for 2 min then supernatant was passed through a 0.2 μm nylonfilter (VWR, PA). For antibiosis assays, 100 μl of an overnight culturefor each plant pathogen was spread onto TSA plates (Thermo Scientific,N.Y.) separately then sterile cork borers (10 mm diameter) were used tobore wells in agar plates. Filtered supernatant of AP193 and its threemutants were separately added to fill wells. Plates were allowed to dryand then incubated at 30° C. overnight. Zones of inhibition weremeasured and compared between mutants and wild-type strain AP193 todetermine their antimicrobial activities against plant pathogens.

LC-MS analysis of bacterial supernatants. Bacterial cultures were grownin 2 ml TSB for 72 hours and then cells were removed by centrifugationat 10,000 x g for 10 min, followed by 0.2 μm filtration of the culturesupernatant. Samples were analyzed by direct injection from m/z 50-1200on a ultra-high pressure liquid chromatography/QTof-mass spectrometer(Waters Acquity UPLC and Q-Tof Premier, Milford, MA) operated at a sprayvoltage of 3.03 kv and the source temperature of 100° C. The MS analysiswas conducted in negative ion mode with a mobile phase of 95%acetonitrile, 5% water and 0.1% formic acid.

In vivo antibiosis of strain AP193 and its mutants against a plantpathogen. Rutgers tomato seeds (Park Seed, USA) were sown in Styrofoamtrays. Three weeks after planting, seedlings were transplanted into a4.5 inch square pot with commercial potting substrate (Sunshine mix, SunGro Horticulture, Agawam, Maine). Three days after transplanting, plantswere sprayed with sterile water or PGPR cell suspensions (10⁶ CFU/ml)that had been washed three times prior to being resuspended in sterilewater and normalized at an OD₆₀₀=1.0 before being serially diluted.PGPR-inoculated plants were placed into a dew chamber at 100% humidityin the dark for two days at 24° C. then transferred to the greenhouse.One day later, plants were challenge-inoculated with X. axonopodis pv.vesicatoria by spraying approximately 10 ml of a 10⁷ CFU/ml pathogensuspension over each plant. Pathogen-inoculated plants were placed inthe dew chamber for two days then placed in the greenhouse. Plants werewatered once daily. Disease severity ratings and harvest were conductedafter 14 days of challenge-inoculation. For disease severity rating,four compound leafs were selected from the bottom of each plant. Thedisease severity of each of the compound leaves was determined by ratingthe disease severity of each leaflet and calculating the average ratingfor the compound leaf. Leaflets were rated using a 0-4 rating scale,where 0=healthy leaflet, 1=<20% necrotic area of the leaflet, 2=20-50%necrotic area of the leaflet, 3=51-80% necrotic area of the leaflet,4=80-100% necrotic area of the leaflet. In addition, dry shoot and rootweights were determined. The experimental design was a randomizedcomplete block with ten replications per treatment. The experiment wasconducted twice.

Data analysis. All data were analyzed by an analysis of variance(ANOVA), and the treatment means were separated by using Fisher'sprotected least significant difference (LSD) test at P=0.05 using SAS9.3 (SAS Institute, Gary, N.C., USA).

Results

Genome Statistics and genetic relatedness of Bacillus species. Genomesequences of 12 different PGPR Bacillus spp. strains were determinedusing next-generation sequencing. The summary statistics for eachBacillus spp. genome sequences and their assemblies are presented inTable 2. The approximate sizes of Bacillus spp. genomes ranged from2.95-4.43 Mbp with an average genome size of 3.93 Mbp, which is similarto the 4.09 Mbp average genome size of complete B. subtilis genomesavailable in GenBank (April, 2015). The percent G+C content of the 12PGPR Bacillus spp. strains ranged from 41.3-46.6%, averaging 45.15% ,which is similar to the average percent G+C content of the B. subtilisgenome sequences available in GenBank (43.72%) (March, 2015). Pairwiseaverage nucleotide identities (ANI), a newly proposed standard forspecies definition in prokaryotes (Richter and Rosselló-Móra, 2009),were calculated for 13 Bacillus PGPR strains to determine theirinterspecies relatedness among Bacillus species. The ANI values for PGPRBacillus spp. strains AB01, AP71, AP79, AP143, AP193, and GB03 againstB. amyloliquefaciens FZB42 (Chen et al., 2007a) were greater than 98%(data not shown), indicating that these PGPR strains are affiliated withthe B. amyloliquefaciens species. The 98.88% ANI of PGPR strain AP254 toB. subtilis subsp. subtilis strain 168 suggests that AP254 is affiliatedwith B. subtilis (data not shown). The pairwise ANI comparison of PGPRstrains INR7, KCTC 3706T, KCTC 13613T, KCTC 13918T, and KCTC 13622Tagainst each other produce ANI values less than 95% (data not shown)suggests that they are distantly related to each other and representdiverse Bacillus species.

Phylogenetic relationship of Bacillus strains. A phylogenetic analysisbased on gyrB gene sequences showed sufficient resolution among Bacillustaxa and was consistent with ANI comparisons. Strains AP71, AP79, AP143,AP193, AB01, and GB03 were grouped together with reference strains of B.amyloliquefaciens subsp. plantarum with high bootstrap support,indicating that they are affiliated with subsp. plantarum. The threestrains of B. amyloliquefaciens subsp. amyloliquefaciens DSM7, TA208,and LL3 clustered as a single clade, separated from strains of subsp.plantarum, supporting the division of two subspecies in B.amyloliquefaciens (Borriss et al., 2011). The placement of strain AP254with B. subtilis subsp. subtilis strain 168 as a single clade withstrong bootstrap support suggests its affiliation with members of the B.subtilis group. (See Hossain et al., Frontiers Plant Science 2015, FIG.1A). A gyrB gene based phylogenetic tree constructed using MaximumLikelihood (ML) methods was also concordant with the phylogenyconstructed using Neighbor-Joining methods (data not shown). In additionto the gyrB-based phylogeny, we constructed a phylogenomic tree using729,383 bp of core genome sequences present within the genome of 25 B.subtilis group isolates to provide a more refined phylogenetic placementof PGPR strains. The topology and allocation of strains to clades in thegyrB phylogeny was similar to the phylogenomic tree (See Hossain et al.,Frontiers Plant Science 2015, FIG. 1B). One notable difference is thatthe topology of the tree regarding the position of strain B. siamensisKCTC13613 differs significantly between the gyrB-based tree and thephylogenomic tree, with the gyrB based phylogeny placing KCTC13613 in aseparate clade whereas the phylogenomic tree included it within amonophyletic group that includes strains of B. amyloliquefaciens subsp.plantarum.

BLAST matrix. Genome wide proteome comparisons of 13 PGPR Bacillusstrains using an all-against-all BLASTp approach demonstrated that PGPRBacillus spp. strains are highly diverse, as indicated by gene familysimilarity between PGPR Bacillus spp. genomes ranging from 32-90% (datanot shown). Consistent with the phylogenetic analysis, high similaritywas found among strains AP71, AP79, AP193, AB01, GB03, and FZB42, withproteomic similarity ranging from 70-90%.

Core-genome analysis. Analysis of genome sequence alignment usingprogressive Mauve determined that the core genome of 13 PGPR Bacillusspp. strains contains 1,407,980 bp of genomic DNA which encode 1,454ORFs (data not shown). Comparison of core genome sequences of the genusBacillus, subgroup B. subtilis, species B. amyloliquefaciens, andsubspecies plantarum demonstrated that as the number of genomesincreases, the number of different subsystems within each respectivecore genome decreases. (See Hossain et al., Frontiers Plant Science2015, FIG. 2A-D). The highest numbers of subsystems in each of the coregenome categories, except for the genus Bacillus core genome, wasdevoted to carbohydrate metabolism. These findings suggest that strainsfrom the genus Bacillus use diverse carbon sources. In addition, thecore genome for the genus Bacillus has more subsystems devoted to RNA,DNA, and protein metabolism compared to carbohydrate metabolism. (SeeHossain et al., Frontiers Plant Science 2015, FIG. 2A-D).

The genome alignment from 28 different subsp. plantarum strains,including six subsp. plantarum strains sequenced in this Example,identified 2,550,854 bp of core genome sequence that is predicted toencode 2,839 ORFs. The genome alignment of 32 B. amyloliquefaciensstrains, including 28 subsp. plantarum strains, identified 2,418,042 bpof core genome sequence predicted to encode 2,773 ORFs.

The genome alignment of 53 strains of B. subtilis group, including the12 strains sequenced in this Example, identified 578,872 bp of coregenome sequence predicted to encode 674 ORFs. The number of proteincoding genes present within the genome of Bacillus spp. (˜4,000) and thelow number of ORFs (674) encoded by their core genomes suggests a largeamount of genomic plasticity among Bacillus genomes that experiencefrequent gene acquisitions and losses. It was observed that the B.amyloliquefaciens core genome was devoid of mobile genetic elements,such as prophages, transposable elements, and plasmids (data not shown).Furthermore, the B. subtilis core genome was also devoid of genes orgenetic clusters linked with iron acquisition and metabolism, secondarymetabolite biosynthesis, signal transduction and phosphorus metabolism.(See Hossain et al., Frontiers Plant Science 2015, FIG. 2A-D).

In this Example, the genus Bacillus core genome was also determined byanalyzing all complete genome sequences from the genus Bacilluscurrently available in GenBank. We determined that the genus Bacilluscontains 194,686 bp of core sequence predicted to encode 201 differentORFs. The predicted functions present in all Bacillus strains arelimited to the following subsystem features: cofactor synthesis, vitaminsynthesis, prosthetic groups and pigments biogenesis, cell wall andcapsule biogenesis, membrane transport, RNA metabolism, nucleosidemetabolism, protein metabolism, regulation and cell signaling, DNAmetabolism, respiration, amino acids and derivatives, sulfur metabolism,and carbohydrate utilization

Comparative analysis of core genes uniquely present in B.amyloliquefaciens subsp. plantarum. Comparison of PGPR-specific genomeswith that of non-PGPR B. subtilis subsp. subtilis str. 168 did notidentify any genes other than essential housekeeping genes that wereconserved within the genomes of PGPR strains (data not shown).Comparative analysis of core genomes from 28 B. amyloliquefaciens subsp.plantarum and 32 B. amyloliquefaciens species identified 193,952 bp ofsequences that are present within the subsp. plantarum core genome butabsent in the B. amyloliquefaciens core genome. Among these genetic locithere were 73 genes shared by all 28 plantarum strains but were notpresent in any strains of subsp. amyloliquefaciens. The putativefunctions of these genes includes transportation (7 genes), regulation(7 genes), signaling (1 gene), carbon degradation (10 genes), synthesisof secondary metabolites (19 genes), and hypothetical proteins (12genes). (See Hossain et al., Frontiers Plant Science 2015, FIG. 2D).Some of these gene products may be involved in interactions with plantsand rhizosphere competence of subsp. plantarum strains (e.g., pectinutilization). For instance, genes required for uptake and use ofD-galacturonate and D-glucuronate are shared among genomes of B.amyloliquefaciens subsp. plantarum strains. These include uxuA(mannonate dehydratase (EC 4.2.1.8)), kdgA (4-hydroxy-2-oxoglutaratealdolase (EC 4.1.3.16)), kdgK (2-dehydro-3-deoxygluconate kinase (EC2.7.1.45)), exuT (hexuronate transporter), exuR (hexuronate utilizationoperon transcriptional repressor), and uxuB (D-mannonate oxidoreductase(EC 1.1.1.57)). In addition, genes required for biosynthesis of thepolyketides difficidin and macrolactin were consistently found in PGPRsubsp. plantarum strains, suggesting their relevance in the biocontrolactivities of these strains.

Gene clusters encoding secondary metabolite biosynthesis and naturalcompetency in strain AP193. Due to our observations of beneficialinteractions between PGPR strain AP193 and both plant and animal hosts(Ran et al., 2012), we selected this strain for more intensive genomeanalysis. Assembly of strain AP193 genome sequences de novo resulted in152 contigs larger than 1 kb, with a combined length of 4,121,826 bp.Analysis of AP193 contig sequences, using the antiSMASH secondarymetabolite prediction program, suggests that gene clusters were presentthat are responsible for synthesis of three different polyketides:bacillaene, macrolactin and difficidin. In order to provide completesequences for these biosynthesis pathways, the gaps between contigs 5and 6, contigs 33 and 38, as well as contigs 27 and 28 were filled usingPCR, followed by DNA sequencing. Each of the gene clusters in AP193 arecollinear to their counterparts in B. amyloliquefaciens FZB42; anaturally competent plant root-colonizing B. amyloliquefaciens isolatewith the ability to promote plant growth and suppress plant pathogens(Chen et al., 2007a). The percent amino acid identities of the proteinsencoded by those clusters were within the range of 98-100% when comparedwith those of FZB42. Secondary metabolite biosynthesis gene clustersinvolved in non-ribosomal synthesis of cyclic lipopeptides surfactins,fengycin and bacillomycin D and of the antimicrobial dipeptide bacilysinpresent in FZB42 were also detected in the AP193 genome. The percentamino acid identities of the AP193 proteins encoded on those clusters tothe FZB42 homologs ranged from 98% to 100%. The lack of naturalcompetency of the PGPR strain AP193 prompted us to determine thepresence of competence-related genes within this strain. We searched theAP193 genome sequences for the presence of competence related genesfound within the genome of FZB42, and observed that all of the genesrequired for encoding the structural components of the competence systemfound in strain FZB42 are present within the genome of AP193 with 98 to100% identity (data not shown); however, genes comQ, comX, and comP areinvolved in regulating quorum-sensing in B. amyloliquefaciens FZB42(Chen et al., 2007a) were absent within the genome of strain AP193 (datanot shown). The absence of comQ, comX, and comP may be responsible forthe lack of natural competency for strain AP193.

AP193 secondary metabolites inhibit the growth of multiple bacterialplant pathogens in vitro. Antimicrobial activities of strain AP193 andits mutants AP193ΔdfnD (deficient in the production of difficidin),AP193ΔsrfAA (deficient in surfactin production), and AP193Δsfp (unableto produce polyketide or lipopepetide due to a deletion of sfp geneencoding 4′-phosphopantetheinyl transferase) were tested against plantpathogens Pseudomonas syringe pv. tabaci, Rhizobium radiobacter,Xanthomonas axonopodis pv. vesicatoria, and Xanthomonas axonopodis pv.campestris. The AP193 wild type strain demonstrated strong antimicrobialactivity, whereas the AP193Δsfp mutant was devoid of an inhibitoryeffect against those plant pathogens. (See Hossain et al., FrontiersPlant Science 2015, FIG. 3), underlining the contribution oflipopeptides and/or polyketides in the bioactivity of AP193. This alsoindicates that the dipeptide bacilysin, whose synthesis is independentof Sfp, was not involved in antagonistic activity expressed in vitro.The AP193ΔsrfAA mutant conferred antimicrobial activity similar towild-type to P. syringe pv. tabaci, R. radiobacter, X. axonopodis pv.vesicatoria, and X. axonopodis pv. Campestris (see

Hossain et al., Frontiers Plant Science 2015, FIG. 3), suggesting thatsurfactin has no putative role in the antibacterial activity of AP193against those plant pathogens under the conditions tested in thisExample. These findings also demonstrated that surfactin neitherinfluences the antimicrobial compound biosynthesis in AP193 nor does itinhibit antibacterial activities of the antibacterial compounds producedby AP193. Difficidin acts as the major antibiotic in antagonism of AP193against plant pathogens P. syringe pv. tabaci, R. radiobacter, X.axonopodis pv. vesicatoria, and X. axonopodis pv. campestris asindicated by the lack of the inhibitory effect of the AP193ΔdfnD mutantagainst those plant pathogens. (See Hossain et al., Frontiers PlantScience 2015, FIG. 4).

We further confirmed that the AP193ΔdfnD and Δsfp mutants lackedsynthesis of difficidin by conducting LC-MS analysis of the cell-freeTSB culture supernatants from wild-type AP193 and each of these mutants.As reported previously, only the deprotonated form of oxydifficidin wasdetectable in bacterial supernatants using MS in the negative mode([M−H]⁻=559.3) (Chen et al., 2006), with a molecular mass of 559.3detected in supernatants of the wild-type AP193 culture but not observedfrom the culture of the ΔdfnD mutant. (See Hossain et al., FrontiersPlant Science 2015, FIG. 4) or from the Δsfp mutant (data not shown).The ΔsrfAA mutant exhibited difficidin synthesis as in the wild-typeAP193 culture (data not shown). These findings demonstrate theimportance of difficidin in the biocontrol activity of subsp. plantarumstrains against plant pathogens.

Strain AP193 secondary metabolites control bacterial spot caused by X.axonopodis pv. vesicatoria in tomato plants. To determine the role ofbioactive compounds produced by strain AP193 in providing protectionagainst plant diseases, the AP193 wild-type strain and its AP193ΔdfnD,AP193Δsfp and AP193ΔsrfAA mutants were applied to tomato plants severaldays before those plants were subsequently inoculated with plantpathogen X. axonopodis pv. vesicatoria. Both AP193 wild-type andAP193ΔsrfAA significantly (P<0.05) reduced disease severity of bacterialspot on tomato plants compared to the disease control (Table 3).Additionally, the application of strain AP193 significantly increasedthe root dry weight of the plants (Table 3). Unlike AP193 wild-type andits AP193ΔsrfAA mutant, strains AP193Δsfp and AP193ΔdfnD neitherprotected tomato plants from severe bacterial spot caused by X.axonopodis pv. vesicatoria nor improved plant growth (Table 3), furthersupporting the importance of difficidin for plant disease protection.These findings are in agreement with the in vitro antibiosis pattern ofAP193 wild-type strain and its AP193ΔdfnD, AP193Δsfp and AP193ΔsrfAAmutants demonstrated against plant pathogen X. axonopodis pv.vesicatoria.

Discussion

PGPR Bacillus spp. strains are used worldwide to improve crop yields andto protect against plant diseases. In this Example, 12 PGPR genomes weresequenced, including B. subtilis, B. pumilus, B. amyloliquefaciens, B.mojavensis, B. siamensis, B. sonorensis, and B. tequilensis. These datawere analyzed using ANI, gyrB-based phylogenies and core genome-basedphylogenies to resolve taxonomic affiliation of Bacillus spp. strains.Our findings demonstrate that half of the strains sequenced in thisExample are affiliated with B. amyloliquefaciens subsp. plantarum,including strain GB03 that was formerly designated as B. subtilis.Previously, B. siamensis type strain KCTC 13613T was proposed as a novelspecies (Sumpavapol et al., 2010), but a Bacillus core genome-basedphylogenomic analysis. (See Hossain et al., Frontiers Plant Science2015, FIG. 1) revealed that B. siamensis KCTC 13613T is insteadaffiliated with B. amyloliquefaciens subsp. plantarum. This findingsupports the results of Jeong et al (Jeong et al., 2012) that determinedthe close affiliation of B. siamensis type strain KCTC 13613T to B.amyloliquefaciens subsp. plantarum based on ANI. These findings alsosupport the continued use of core genome-based phylogenomic approachesto provide better phylogenetic resolution than analyses that use asingle housekeeping gene (e.g., gyrB). Phylogenies based on gyrB andcore genome sequences demonstrate that B. amyloliquefaciens subsp.plantarum are highly similar, but comparison of their proteomesdemonstrates that they are closely related, yet distinct, and may exertplant growth-promoting activities through different mechanisms.

B. amyloliquefaciens subsp. plantarum strain ABO1 was isolated from theintestine of channel catfish (Ran et al., 2012), but its affiliationwith plant-associated strains may suggest transient presence within afish gastrointestinal tract; however, given that the fish feed issoy-based it is likely that the plant-based diet was also a factor inthe growth of this strain within a fish intestine. Similarly, B.siamensis type strain KCTC 13613T was found to be closely affiliatedwith B. amyloliquefaciens subsp. plantarum and was isolated from saltedcrab, rather than a plant-associated source. The efficacy of strainsAB01, AP193, and other plant-associated strains as probiotics in fishshows the capacity for biocontrol of animal and plant pathogens as wellas an overlap in host colonization (Ran et al., 2012).

With rapid advances in sequencing technologies it is now possible toextend genomic analysis beyond individual genomes to analyze coregenomes (Medini et al., 2008). In this Example, core genomic analyseswere conducted on PGPR strains from species affiliated with the B.subtilis group. This analysis identified 73 genes exclusively presentamong all subsp. plantarum that are absent in subsp. amyloliquefaciensstrains. This small number of subsp. plantarum-specific genes agreeswith a previous report that identified 130 subsp. plantarum-specificgenes using a limited number of genome sequences from subsp. plantarumstrains (He et al., 2012). Of these 73 plantarum-specific genesidentified in this Example, many are predicted to be important forplant-associated and soil-associated functions. For example, genes thatare required for the use of D-galacturonate and D-glucuronate were foundin the pool of B. amyloliquefaciens subsp. plantarum-specific coregenes. This observation is consistent with the absence of these genes inthe genome of B. amyloliquefaciens subsp. amyloliquefaciens DSM7(Ruckert et al., 2011), a strain without any reported PGPRactivity.Pectin, a complex polymer found in plant tissues, is brokendown to D-glucuronate and D-galacturonate which then serves as a carbonsource for bacterial growth (Nemoz et al., 1976). This pectin couldpotentially serve as a nutrient source for efficient root colonizationof PGPR through competitive nutrient uptake. Therefore, the presence ofgenes that enable D-galacturonate and D-glucuronate utilization could beadvantageous for B. amyloliquefaciens subsp. plantarum for plantgrowth-promoting activity through efficient root colonization.

Since many of the PGPR strains are from the B. subtilis group, the coregenome estimation was expanded to include a larger number of B. subtilisstrains. Increasing the number of Bacillus subtilis genomes analyzed to53 resulted in a 579,166 bp core genome that is predicted to encode 674ORFs. This smaller number of predicted genes reflects genomic diversityamong the B. subtilis group. This finding demonstrates that the numberof ORFs found in the B. subtilis group core genome is close to thenumber of B. subtilis ORFs that are considered as indispensable forgrowth in complex media (610 ORFs)(http://www.minibacilius.org/project#genes).

To validate a gene's involvement in plant-related processes, it isessential to construct isogenic mutants that are devoid of those genes.Therefore, we deleted genes from PGPR strain AP193 to evaluate the roleof secondary metabolite biosynthesis gene clusters in the biologicalcontrol of plant pathogens. To do this, a methylated shuttle vectorpNZT1 (Zakataeva et al., 2010) with gene deletion constructs deliveredtargeted genetic modifications to AP193, demonstrating the efficacy ofin vitro methylation of plasmids by cell-free extract in circumventing arestriction system that was presumed to have prevented transformationthrough electroporation.

Difficidin is a highly unsaturated 22-membered macrocylic polyenelactone phosphate ester with broad-spectrum antibacterial activity(Zimmerman et A, 1987). Difficidin expressed by strain FZB42, togetherwith the dipeptide bacilysin, are antagonistic against Erwiniaamylovora—the causative agent of fire blight disease in orchard trees(Chen et al.. 2009). This Example using an isogenic mutant AP193ΔdfnDdemonstrated for the first time that difficidin solely, not inconjunction with any other polyketides or dipeptides, exerts in vitroantibacterial activity against plant pathogens, such as Pseudomonassyringe pv. tabaci, Rhizobium radiobacter, Xanthomonas axonopodis pv.vesicatoria and Xanthomonas axonopodis pv. campestris. We alsodemonstrated, by using isogenic mutant AP193ΔdfnD, that difficidinexpression is responsible for control of bacterial spot disease intomato plants caused by X. axonopodis pv. vesicatoria. Taken together,these findings demonstrate that difficidin is the most important strainAP193 secondary metabolite for biological control of plant diseases dueto bacterial pathogens. In addition, the construction of the sfp genedeletion allowed investigation of multiple secondary metabolitesproduced by AP193 and their individual contributions to biocontrolactivity. The sfp deletion mutant lost antagonistic activity againsteach pathogen that was susceptible to the AP193 wild-type strain.Mutants with the sfp deletion are expected to lose the ability tosynthesize difficidin in addition to other metabolites. Because the lackof antimicrobial activity of AP193Δsfp is consistent with that of theAP193ΔdfnD mutant, this therefore suggests that difficidin is theprimary metabolite responsible for in vitro inhibition of bacterialpathogens. In contrast, the surfactin mutant retained antimicrobialactivity against all plant pathogens tested, demonstrating thatsurfactin is neither critical for in vitro antibiotic activity norinfluences the synthesis or secretion of other secondary metabolitebiosynthesis in this Bacillus spp. strain; however, surfactin mayinfluence plant growth promoting activity since it has been observedthat surfactin of B. subtilis elicits ISR in plants (Ongena et al.,2007) and is expressed in the plant cells colonized by FZB42 (Fan etal., 2011).

By studying the contributions of genetic loci that are conserved amongtop-performing PGPR strains we continue to uncover the relativecontributions of genes in plant colonization, growth promotion, and/orpathogen biocontrol. In particular, future investigation of genesrelated to the uptake and use of pectin-derived sugars will helpdetermine the relative importance of these genes for colonization ofplants and persistence within this microbiome. Comparative genomicanalysis of Bacillus spp. PGPR strains has led to a better understandingof gene products and provides a foundation to develop applicationstrategies that result in greater plant growth promotion and biocontrolactivity.

Tables

TABLE 1 Bacterial strains and plasmids used in this Example. Strains orplasmids Relevant characteristics Source or reference E. coli K12 ER2925dcm-6 dam13::Tn9 New England Biolabs B. amyloliquefaciens subsp. Wildtype Dr. Joseph Kloepper plantarum strain AP193 (Department ofEntomology and Plant Pathology, Auburn University) AP193Δsfp deficientin lipopeptides This study and polyketides AP193ΔsrfAA deficient insurfactin This study production AP193ΔdfnD deficient in difficidin Thisstudy production Bacillus amyloliquefaciens Wild type (Chen et al.,2007b) FZB42 pMK4 E. coli-Bacillus shuttle BGSC plasmid, rolling circlereplicative, Cm^(R) pNZT1 Replication Xiaozhou Zhang, Virginiathermosensitive derivative Tech of the rolling-circle plasmid pWV01 (pG⁺replicon, Em^(R)) pNZ-sfp pNZT1 with upstream and This study downstreamsequences of gene sfp pNZ-srf pNZT1 with knock-out This study constructof srfAA pNZ-dif pNZT1 with knock-out This study construct of dfnD

TABLE 2 Summary of draft genomes of Bacillus species sequenced used inthis Example Number of Size (total NCBI NCBI Short Approx. Number ofContigs bp in BioProject Read Archive sequence predicted Isolates (>1kb) assembly) % G + C Number Accession No. coverage (x) ORFs AB01 203,903,296 46.4 PRJNA239317 SRX475739 44 3944 AP71 198 4,278,192 45.7PRJNA239317 SRX475740 15 4531 AP79 47 4,236,770 45.8 PRJNA239317SRX475741 31 4368 AP143 146 2,956,670 46.6 PRJNA239317 SRX475742 24 3324AP193 152 4,121,826 46.3 PRJNA239317 SRX475807 37 4159 AP254 594,048,419 43.8 PRJNA239317 SRX475808 29 4717 GB03 26 3,849,547 46.5PRJNA227787 SRX380920 560 3928 INR7 44 3,681,709 41.3 PRJNA227786SRX447924 750 3857 KCTC 3706T 17 3,935,582 43.7 PRJNA227789 SRX447926895 4140 KCTC 13613T 23 3,779,696 46.3 PRJNA161489 SRX450083 500 3915KCTC 13918T 32 4,428,962 45.5 PRJNA227788 SRX450084 1000 4704 KCTC13622T 33 3,981,302 43.9 PRJNA227791 SRX450086 1000 4299

TABLE 3 Effects of plant growth-promoting rhizobacteria (PGPR) strainson severity of bacterial spot disease and plant growth Root DryStrain^(ab) Disease severity^(c) Shoot Dry Weight (g) Weight (g) DiseaseControl 2.11a 2.07bc 0.378c AP193 1.30b 2.18b 0.453a AP193ΔsrfAA 1.48b2.16b 0.423abc AP193Δsfp 2.31a 2.18b 0.405abc AP193Δdif 2.06a 2.00c0.389bc Healthy Control 0.00c 2.38a 0.435ab LSD 0.35 0.15 0.050 Note:^(a)The experimental design was a randomized complete block with tenreplications per treatment. The experiment was conducted twice. Valuesfollowed by the same letter were not significantly different (P = 0.05)according to Fischer's protected LSD. ^(b)One plant was in eachreplication. Plants were sprayed with PGPR suspension (10⁶ CFU/ml) oneweek after transplanting, and were challenge-inoculated with pathogensolutions (10⁷ CFU/ml) three days after inoculating PGPR. ^(c)Diseaseseverity ratings and harvest were done 14 days later. For diseaseseverity rating, four compound leafs were selected from the bottom ofeach plant. The disease severity of each of the compound leaves wasdetermined by rating the disease severity of each leaflet andcalculating the average rating for the compound leaf. The leaflet wasrated using a 0-4 rating scale, where 0 = healthy leaflet, 1 = <20%necrotic area of the leaflet, 2 = 20-50% necrotic area of the leaflet, 3= 51-80% necrotic area of the leaflet, 4 = 80-100% necrotic area of theleaflet, or fully dead leaflet.

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Ryu, C.-M., Farag, M. A., Hu, C.-H., Reddy, M. S., Kloepper, J. W., andParé, P. W. (2004). Bacterial Volatiles Induce Systemic Resistance inArabidopsis. Plant Physiol 134, 1017-1026. doi: 10.1104/pp.103.026583.

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Example 2—Efficiency of Pectin Supplement in Root Colonization and PlantGrowth-Promotion of Soybean Plants by Bacillus amyloliquefaciens subsp.plantarum (Bap)

Abstract

A second greenhouse experiment was conducted to determine the effects ofpectin on root colonization, nodulation by indigenous soil rhizobia, andplant growth-promotion activity of Bacillus amyloliquefaciens subsp.plantarum (Bap) rifR strains AP193 and AP143 on soybean. The overallhypothesis tested is that the complex carbohydrate pectin will enhanceBap-mediated plant growth promotion. The experimental design includedsoybean seeds planted in soil that contained Bap strains with or without0.1% pectin supplement, along with a water control, and after 28 daysthe plants were harvested and analyzed for statistical significance. Thedata indicate that Bap rifR strains with pectin supplement enhancedsoybean shoot and root length in greenhouse conditions. Dry shoot androot weights of Bap rifR strains with pectin supplement also increased,but were not significantly different compared to Bap rifR strainswithout pectin supplement. Bap rifR strains with pectin supplementincreased significantly root nodulation of soybean. However,rhizobacterial populations recovered from the rhizo sphere of soybeanplant roots were not significantly different.

Materials and Methods

Preparation of rhizobacterial cells. Plant growth-promotingrhizobacteria (PGPR) Bacillus amyloliquefaciens subsp. plantarum (Bap)strains AP193 and AP143 were selected based on their capacity forutilization of pectin as a C and energy source. Bacillus thuringiensissubsp. kurstaki (Btk) strain HD73 was collected from the USDA-ARSculture collection (Ames, Iowa) that was selected for inability todegrade and use pectin.

Three bacterial strains were cultured on TSA (BD Difco™ Agar, USA) with50 μg/m1 Rifampicin (Sigma, USA) from the cryo stocks (−80 ° C.) at 28°C. for 24 hours. A single colony of each strain was inoculated in 50 m1tubes containing 35 ml TSB with rifampicin (50 μg/ml) in tubes wrappedwith aluminum foil to avoid light. Bacterial cultures were incubated for48 hours with shaking at 220 rpm at 30° C. The bacterial cells were thenspun down using Sorvall Legend RT centrifuge (The Thermo Scientific,USA) at 10,000×g for 10 minutes and the supernatant was discarded, andthe pellet was washed with sterile water to remove the media. After thefinal wash, the supernatant was discarded and re-suspended in sterileMilli-Q water. The mid log phase bacterial suspension optical densitywas calculated at on optical density of 600 nm (OD600) using a GENESYS™10S UV-Vis Spectrophotometer (Thermo Scientific, USA). The OD600 of eachrhizobacterial strain was serially diluted for calculated ofcolony-forming units (CFU) per milliliter. Suspensions were diluted toobtain 1×106 CFU/ml for inoculation of soybean seeds.

Soil preparation and pectin mixture. Field soil sieved to remove rootdebris was used for the greenhouse experiment. Soil (450 g) was placedin each cone-tainer tube (lightweight large Deepots D40L, Stuewe & Sons,Danville, Ill., USA) that contained three cotton balls in the bottom toretain soil. Pectin power (EC No. 232-553-0, Tokyo chemical industryco., Toshima, Kita-Ku, Tokyo, Japan) from citrus source was mixedthoroughly with field soil using soil mixing machine at a rate of 1 gper 1,000 g field soil.

Soybean seed inoculation and Plant growth measurement. Soybean seed(‘Asgrow 6702 RR’) not treated with chemicals was used as in theprevious experiment. One seed was placed in each cone-tainer, and 1 mlof 106 CFU was pipetted over each seed. Then, 5 g of soil was placedover each seed. Each cone-tainer rack was covered by plastic sheet toprevent soil desiccation for 48 hours. Afterwards, cone-tainer rackswere transferred to the greenhouse chamber and tubes were watered twicedaily.

After 28 days, all the plants were harvested for plant morphometricmeasurement. Shoot length was measured from the growing apical region tothe basal region connected to the root. Root nodules were visuallycounted from all plants. The soil (approximately 448 g) was removedgently from the roots of each plant and placed in a 15 ml falcon tubes.Nine ml sterile water were added to each conical tube, and tubes werevortexed thoroughly. Then serial dilutions were made from 1:10 to 1:1000in sterile Milli-Q water in microcentrifuge tubes and plated on TSAplates with rifampicin (50 ⊐ g/ml) for each dilution and incubated at28° C. for 24 to 48 hours. Colonies that grew on the rif TSA plates werecounted and expressed in log10 CFU/ml. For root dry mass measurement,roots were washed to remove soil and dried in oven 45° C. After washingroots to remove all soil, roots and shoots were dried in an oven at 45°C. for dry weights. Root length was measured from the root apex toroot-stem junction.

Experimental design and statistical data analysis. Cone-tainers werearranged in a completely randomized design (RCD) with 8 treatments andwith 12 replications, with each replication being a single plant in asingle cone-tainer. The data of shoot height, root height, dry shootweight, dry root weight, root nodules, and rhizobacterial growth wereanalyzed with SAS 9.4 software (SAS Institute, Cary, N.C.) using theproc GLIMMIX. Each treatment means was compared using LSMEANS at P=0.05level of significance.

Results

The effects of Bap strains AP193 and AP143 on shoot length, root length,dry shoot weight, dry root weight, and root nodulation of soybean variedwith pectin amendment are shown in Table 4. Each experimental controland treatment had 12 replicates, except for AP143 treatment which had 11replicates due to lack of germination.

TABLE 4 Effect of pectin amendment on soybean growth, nodulation byindigenous rhizobia, and root colonization by Bap strains and Btk. Plantgrowth metrics Mean no. of Shoot Root Dry Shoot Dry Root nodulation byRoot length length weight weight indigenous soil colonization Treatment(cm) (cm) (g) (g) rhizobia (Log CFU/g) Water control 28.71c 18.58b 0.33d0.07cd 0 0 Pectin control 38.04b 24.25a 0.39bc 0.07cd 5.41ab 2.4c AP193W33.91b 25.29a 0.42bc 0.10bc 1.50bcd 4.48a AP193P 46.37a 26.41a 0.64a0.15ab 7.33a 4.94a AP143W 36.80b 22.89a 0.45b 0.16a 2.16bc 3.76b AP143P43.12ab 22.79ab 0.59ab 0.15ab 9.33a 4.10ab HD73W 30.37bc 18.08b 0.35cd0.06cd 0.66cd 2.03c HD73P 36.70b 23.95a 0.35cd 0.06d 0.91cd 1.43c *Bap^(x) stain = AP193 & AP143; Btk^(y) strain = HD73 * Mean values fromthe greenhouse experiment with eight treatments and 12 replications.Mean value in the column followed by the same letter are notsignificantly different at P ≤ 0.05 using Tukey's multiple comparisontests.

The shoot length was significantly enhanced by pectin supplement andinoculation with Bap strain AP193 but not by Bap strain AP143 withpectin supplement. The mean shoot length of Bap strains AP193 and AP143with pectin supplement were 46.40 cm and 43.10 cm. Shoot of Bap strainAP193 with pectin supplement was higher than Bap strain AP193 withoutpectin supplement. However, with Bap strain AP143 and pectin supplement,mean shoot length was slightly higher than treatment without pectinsupplement. Mean root lengths of Bap strains AP193 and AP143 with pectinsupplement were slightly higher but not significantly different comparedto Bap strains AP193 and AP143 without pectin supplement.

Pectin amendment did not result in significantly higher shoot weightsfor treatment with either of the Bap strains. Dry shoot weight of Bapstrain AP143 with pectin supplement were 0.59 g and not significantlydifferent than Bap strain AP143 without pectin supplement. Soybean dryroot weight of Bap stain AP193 with pectin supplement were 0.15 g and itwas slightly higher than Bap strain AP193 without pectin supplement.There was no significant difference found between Bap strain AP193 withpectin supplement and Bap strain AP193 without pectin supplement.

Nodulation of soybean roots by native soil rhizobia (Table 4) werepresent with application of pectin put not in the control with no pectinand no PGPR strains. Root nodulation by indigenous rhizobia of Bapstrains AP193 and AP143 with pectin supplement were significantlydifferent than Bap strains AP193 and AP143 without pectin supplement.Mean root nodulation by native rhizobia of Bap strains AP193 and AP143with pectin supplement were 7.33 and 9.33. Root nodulation were notobserved in the water control treatment.

The effects of pectin supplement on root colonization by the inoculatedBap strains are shown in Table 4. The rhizosphere bacterial populationsof Bap strain AP143 with pectin supplement were recovered within 24hours after incubation at 28 ° C. Bap strain AP193 with pectinsupplement was recovered within 36 hours after incubation at 28 ° C. Thecolony_morphology of recovered Bap and Btk strains were alike to theapplied Bap and Btk strains. Bap and Btk strains were observed in thepectin control but not the water control. With all three bacteria,supplementation with pectin did not significantly increased rootcolonization.

Discussion

The overall results of the greenhouse experiment support the hypothesisthat pectin supplement enhances plant growth-promotion caused by BapPGPR strains. In this study, greenhouse results showed that Bap strainswith pectin supplement have the ability to enhance soybean plant growthby root colonization without causing damage on the roots, leaves, andshoots. The results (Table 4) showed that plant responses to pectinamendment with and without two Bap strains depended on the strain. Forexample, comparing effect on plants of Bap strain AP143 with and withoutpectin, there was no significant effect on shoot or root length andweights. Bap strain AP193, shoot length and root weight weresignificantly enhanced with pectin amendment, Comparing the effects ofpectin amendments with and without Bap strains on nodulation by nativesoil rhizobia, Bap strain AP143 and pectin had enhanced nodulationcompared to pectin alone, but with Bap strain AP193, there was nosignificant difference in nodulation with and without pectin.

Bap strains AP193 and AP143 soil amended with pectin enhanced soybeanshoot and root growth in greenhouse conditions will be important tounderstanding how the concentration, source, and structure of pectinimpacts the degradation and utilization by rhizobacteria to promoteplant growth. Pectin composition in monocots (2-10%) and dicots (35%)vary widely due to plant primary cell wall structure (Ridley et al.,2001). Recent studies have found that 0.5% pectin increased biofilmformation on Arabidopsis thaliana by Bacillus subtilis, but little isknown about its effect on plant growth (Beauregard et al., 2013).

There have been consistent observations that Bap strains with combinedwith pectin amendment increased the frequency of soybean rootnodulation. Soybean root nodulation in Bap strains AP193 and AP143 withpectin supplement were fivefold and fourfold greater compared to Bapstrains AP193 and AP143 without pectin supplement. This results indicatethat pectin mixed field soil with Bap strain might induce soybeannodulation substantially than Bap strain without pectin supplement.There is a report that mixed inoculation of Bradyrhizobium japonicumwith Bacillus amyloliquefaciens strain LL2012 enhanced soybeannodulation (Masciarelli et al., 2014). Another study also indicated thatBacillus cereus UW85 increased soybean nodulation in a growth chamberand the field conditions without inoculation of Bradyrhizobia spp.(Halverson and Handelsman, 1991). Bradyrhizobia spp. in the field soilsmay have enhanced root nodulation in the presence of Bap strains withpectin supplement. Co-inoculation of Bacillus polymyxa or B. subtiliswith Azospirillum sp. was shown to allowed pectin degradation andnitrogen fixation by using pectin as a sole carbon and energy sources(Khammas, 1992).

Although, Bap strains with pectin supplement populations were notsignificantly different that Bap strains without pectin supplementpopulations, these re-isolations of Bap strains were only conductedusing rhizosphere bacterial population counts. The two BAP strains, thatcolonization, while not significantly different, was about 0.5 loggreater with pectin supplement, so a separate study on the effect ofcolonization at several time point, will be conducted. Therefore, futurestudies need to address whether PGPR bacteria are colonizing on the rootrhizosphere, rhizoplane, or as an endophytic population. It may bepossible that the Bap strains are not increasing in the CFU countswithin the rhizosphere, rhizoplane or endophytically, but are utilizingthe pectin and increasing the production of bioactive metabolites thatare stimulating plant growth. By determining the production ofBap-derived metabolites in these different treatment groups we will beable to test the hypothesis.

REFERENCES

Beauregard, P. B., Chai, Y., Vlamakis, H., Losick, R., Kolter, R., 2013.Bacillus subtilis biofilm induction by plant polysaccharides.Proceedings of the National Academy of Sciences 110, E1621-E1630.

Halverson, L., Handelsman, J., 1991. Enhancement of soybean nodulationby Bacillus cereus UW85 in the field and in a growth chamber. ApplEnviron Microbiol 57, 2767-2770.

Khammas, K. K., P, 1992. Pectin decomposition and associated nitrogenfixation by mixed cultures of Azospirillum and Bacillus species.Canadian journal of microbiology 38, 794-797.

Masciarelli, O., Llanes, A., Luna, V., 2014. A new PGPR co-inoculatedwith Bradyrhizobium japonicum enhances soybean nodulation.Microbiological research 169, 609-615.

Ridley, B. L., O′Neill, M. A., Mohnen, D., 2001. Pectins: structure,biosynthesis, and oligogalacturonide-related signaling. Phytochemistry57, 929-967.

Example 3—Enhanced Soybean Growth Root Nodulation from PGPR and PectinApplication

In recent years, PGPR have been developed as biofertilizers to promoteplant growth (Calvo, 2014; Yan, 2002; Kloepper, 1994) and providebiological control of plant diseases (Liu et al., 2016a & b). co-PIProf. Kloepper has a collection of PGPR strains that have shown plantgrowth-promoting activity in field soils on corn, soybean, wheat,canola, and several vegetable crops including tomato, pepper, andcabbage. Many of these PGPR strains have been identified as Bacillusvelezensis (Note: their phylogenetic affiliation was previouslydescribed as B. amyloliquefaciens subsp. plantarum; Hossain et al.,2015; Bashan et al., 2014). Bacillus spp. strains can induce plantgrowth by diverse mechanisms including solubilizing phosphate andproduction of plant hormones such as indole acetic acid (Niazi et al.,2014). Many PGPR strains also produce many metabolites that protectplants against bacterial and fungal pathogens, or can induce systemicresistance (Adesemoye et al., 2009; Avdeeva et al., 2014, Liu et al.,2016a & b).

Our labs conducted a comparative genomic study on our 28 best-performingB. velezensis (Bv) strains, and found 73 genes consistently present inthese genomes, but not present in other Bacillus species that lackedPGPR activity. These PGPR-specific gene products include the uptake andutilization of pectin-derived sugars (Hossain et al., 2015). Wesubsequently found that all 59 of the B. velezensis strains in our PGPRcollection could use pectin as a carbon and energy source (data notshown). This led to our central hypothesis that pectin amendment to soilwill increase root colonization by By PGPR strains leading to improvedefficacy of plant growth promotion. There are no previous publicationson the use of pectin to enhance rhizobacteria-mediated plant growthpromotion or to induce legume nodulation. Our greenhouse trials to testour central hypothesis showed a significant 2-fold increase in soybeanroot weight when both By and pectin were amended to an agricultural soilcompared to the water control (FIG. 3). Interestingly, we also observedan over 10-fold increase in soybean nodulation (FIG. 4). In addition,the size of the nodules produced in plants that had a PGPR inoculum andpectin supplementation greatly increased compared to a By strain alone(FIG. 4A). We hypothesize that increased pectinolytic activity or otherBy metabolites stimulated by pectin amendment enhances nodulation ofsoybean roots by native Bradyrhizobium japonicum present in soilspreviously cropped with soybean. We predict that the larger and morenumerous nodules present on the more extensive soybean root systems willresult in significant soybean yield increases. The greenhouseexperiments represented in the above figures have been replicated andshowed the same significant increases in root mass and nodulation sizeand frequency when pectin and PGPR were amended to soil (data notshown). These studies provide strong support for extending this researchinto field studies to evaluate soybean yield. We further anticipate thatthe use of pectin and PGPR inoculants will be a cost-effective andsustainable method to promote plant growth and reduce the variabilityinherent in the use of beneficial PGPR strains in field soils.

REFERENCES

Adesemoye A O, Torbert H A, & Kloepper J W. 2009. Plant Growth-PromotingRhizobacteria Allow Reduced Application Rates of Chemical Fertilizers.Microbial Ecology 58:921-929.

Avdeeva L V, Dragovoz I V, Korzh lu V, Leonova N O, Iutinskaia G A,Berezhnaia A V, Kuptsov V N, Mandrik M N, & Kolomiets E I. 2014.Antagonistic activity of Bacillus amyloliquefaciens subsp. plantarum IMVB-7404 and BIM B-439D strains towards pathogenic bacteria andmicromycetes]. Mikrobiol Z. 76:27-33.

Bashan Y, de-Bashan L E, Prabhu S R, & Hernandez J P. 2014. Advances inplant growth-promoting bacterial inoculant technology: formulations andpractical perspectives (1998-2013). Plant Soil. 378 :1-33.

Calvo P, Nelson L, & Kloepper J W.2014. Agricultural uses of plantbiostimulants. Plant Soil. 383:3-41.

Corbin, E J., Brockwell, j., and Gault, R. R. 1977. Nodulation studieson chickpea (Cicer arietinum). Australian Journal of ExperimentalAgriculture and Animal Husbandry, 17: 126-134.

Hassan, M K. 2016. The Role of Pectin Utilization in Root Colonizationand Plant Growth-Promotion by Bacillus amyloliquefaciens subsp.plantarum. Thesis submitted to University of Auburn.

Hossain M J, Ran C, Liu K, Ryu C M, Rasmussen-Ivey C R, Williams M A,Hassan M K, Choi S K, Jeong H, Newman M, Kloepper J W, & Liles M R.2015. Deciphering the conserved genetic loci implicated in plant diseasecontrol through comparative genomics of B. amyloliquefaciens subspplantarum. Frontiers in Plant Science 6.

ISTA. 1993. Proceedings of the International Seed Testing Association,International Rules for Seed Testing. Seed Sci. Technol. 21:25-30.

Liu, K., Garrett, C., Fadamiro, H., Kloepper, J W. 2016a. Antagonism ofblack rot in cabbage by mixtures of plant growth-promoting rhizobacteria(PGPR) BioControl. 61: 605-613.

Liu, K., Garrett, C., Fadamiro, H., Kloepper, J W. 2016b. Induction ofsystemic resistance in Chinese cabbage against black rot by plantgrowth-promoting rhizobacteria. Biological Control. 99: 8-13.

Kjeldahl, J. 1883. Neue Methode zur Bestimmung des Stickstoffs inorganischen Körpern. Zeitschrift für analytische Chemie, 22: 366-383.

Kloepper J W. 1994. Plant-growth-promoting rhizobacteria (othersystems). Azospirillum/plant associations, ed Okon Y (CRC Press, BocaRaton), pp 139-154.

Kloepper J W, Ryu C M, & Zhang S A. 2004. Induced systemic resistanceand promotion of plant growth by Bacillus spp. Phytopathology94:1259-1266.

Kloepper, J W and Schroth, M N. 1981. Development of a powderformulation of rhizobacteria for inoculation of potato seed pieces.Phytopathology 71:590-592.

Niazi, A., Manzoor, S., Asari, S., Bejai, S., Meijer, J.,Bongcam-Rudloff, E. 2014. Genome Analysis of Bacillus amyloliquefaciensSubsp. plantarum UCMB5113: A Rhizobacterium that improves plant growthand stress management. PLOS ONE. 9: e104651.

Yan Z N, Reddy M S, Ryu C M, Mclnroy J A, Wilson M, & Kloepper J W.2002. Induced systemic protection against tomato late blight elicited byplant growth-promoting rhizobacteria. Phytopathology. 92:1329-1333.

Example 4—Grant Proposal for “Use of Pectin to Enhance Efficacy of PlantGrowth-Promoting Rhizobacteria (PGPR) Strains to Improve AgriculturalProductivity”

Introduction

Plant growth-promoting rhizobacteria (PGPR) have been identified thatcontrol plant diseases and promote overall plant growth. “Rhizobacteria”means root-colonizing bacteria, and hence, root colonization isessential for plant growth promotion by PGPR strains. Plant roots exudevarious organic compounds, including sugars, and successful bacterialcolonization hinges on nutrient uptake from the host plants throughextracellular enzymatic activity. Strains of Bacillus amyloliquefacienssubsp. plantarum (Bap) colonize plant roots, and have been used asbiofertilizers or biocontrol agents during the past decades. Some of thebest-performing PGPR Bap strains at Auburn have been subjected to acomparative genomic analysis, which indicates that the use of pectin isa conserved trait among these sequenced strains. As a structuralcomponent of the plant cell wall, much is known regarding pectinbiochemistry and plant synthesis; however, little is known about thepossible role of pectin in root colonization. In fact, the currentscientific paradigm regards pectin utilization as a function expressedby plant pathogens, and not as a potentially useful characteristicexpressed by plant-associated PGPR strains. We now have experimentalevidence that our best-performing PGPR Bap strains can obtain carbon andenergy via 1) production of an extracellular pectinolytic enzyme(s) thatdegrades plant pectin into hexuronate sugars, 2) transport ofpectin-derived sugars, and 3) utilization of these pectin-derived sugarsfor bacterial respiration. While these PGPR Bap strains consistentlyperform well under lab or greenhouse conditions, field trials are morevariable in PGPR efficacy. We hypothesize that supplementing pectinlevels on seeds or in the plant rhizosphere will improve the efficacy ofPGPR strains in stimulating plant growth and disease control.

Introduction

There is a growing need for environmentally sustainable methods topromote plant growth in agriculture that has prompted the search formethods, like the probiotic strategies described in this proposal, tocost-effectively enhance plant growth. Plant growth-promotingrhizobacteria (PGPR) have been developed as biofertilizers to promoteplant growth (8-11). While many species of bacteria are classified asPGPR strains, Bacillus species have been closely studied due to theirspore-forming activity that confers a longer shelf life and greaterviability in commercial formulations. Within this genus, strains ofBacillus amyloliquefaciens subsp. plantarum (Bap) have emerged asespecially effective PGPR strains that lack any potential forpathogenesis (12, 13). At Auburn University, co-PI Kloepper hascollected over 1,000 rhizobacteria isolates, of which over 300 haveshown plant growth promoting activity in field soils on corn, soybeanand wheat plants and 59 of these PGPR strains have been identified asBap strains.

Some PGPR strains, including many Bap strains, can be used asbiostimulants to promote plant growth (14). For example, microbialinoculants can solubilize phosphorus and/or fix nitrogen that can thenbe absorbed by plant roots, directly stimulating plant growth. There isa large body of literature on the use of bacterial inoculants fornitrogen fixation (15), and many Bacillus spp. strains have beenidentified as phosphate-solubilizing bacteria with commercial potentialas biofertilizers (16). In addition, PGPR strains have been found toproduce many secondary metabolites that have antibiotic activity againstbacterial and/or fungal pathogens (17-19), including our discovery ofthe novel and potent antibiotic Bacillusin A produced by a Bap strain(20). PGPR strains can also induce the control of plant disease throughproduction of compounds that induce plant systemic acquired resistance(SAR; mediated by salicylic acid) and induced systemic resistance (ISR;jasmonic acid-dependent) (8, 21, 22).

The earliest reported studies of seed bacterization for agriculturalpurposes dates to the use of Rhizobium inoculants on legumes in 1895(23). Yield increases for cereal crops after bacterial inoculants wereapplied were observed in a variety of Soviet and Indian studiesthroughout the 1960s and early 1970s (23). However, field studies haveconsistently produced lower yields than greenhouse studies, suggestingthat the introduced microbial population declines rapidly after soilinoculation (23, 24). This decline was likely due to an inability of thePGPR strains to compete and thrive within the rhizosphere. As describedby Hawes et al. (26), “Efforts to improve plant health by addingexogenous populations of beneficial microorganisms (biological control)are notoriously unreliable” (25). Despite the inherent difficulties ofusing bacterial inoculants, the North American market for biostimulants,which includes PGPR, is estimated to grow to $490 million by 2018 (26).There is therefore strong interest in strategies that can enhance theefficacy of PGPR strains to improve agricultural productivity.

Our labs have conducted a comparative genomic study on ourbest-performing PGPR Bap strains, and we were able to identify 73 genesthat were consistently present within all 28 genomes surveyed, but notpresent in other strains of that B. amyloliquefaciens were known not tohave PGPR activity (7). Importantly, we found that genes related to theuptake and utilization of pectin-derived sugars were always observedwithin these 28 PGPR Bap strains (7). This led to the hypothesis thatroot-derived pectin is important for beneficial rhizobacteria tocolonize roots, produce bioactive metabolites and provide nutrients toplants, resulting in improved efficacy of plant growth promotion.

There is a significant knowledge base for pectin biochemistry that canbenefit this project. Henri Braconnot discovered pectin in 1825 (27),and pectic substances are now known to be highly complexheteropolysaccharides that comprise a major component of plant primarycell walls in addition to cellulose and hemicellulose (28). For example,the primary cell wall of Sycamore is composed of 34% pectin, 24%hemicellulose, 23% cellulose, and 19% hydroxyproline-rich glycoprotein(29). The highest levels of pectin occur in the fruits, leaves, androots of plants, so this is consistent with the potential for pectin toprovide a needed root-derived nutrient source for beneficialmicroorganisms. Pectin is found in the middle lamella between cells,where it helps to bind cells together, and the availability andstructure of pectins (polygalacturonans) varies among plant species(30). The demethylesterification of pectins, which is mediated throughthe action of pectin methylesterases, is an important process in seedgermination (31) and in release of root border cells (32) which areliving cells programmed to separate from roots into the external soilenvironment (26). Importantly, the mucilage produced by root bordercells is rich in pectin and there is a “dramatic increase in the levelsof soluble, de-esterified pectin in the root tip during border celldevelopment” (32). The pectin-rich mucilage associated with root bordercell release has been shown to be important for root penetration intosoil, as well as binding metal cations such as aluminum to preventtoxicity (33, 34). However, to our knowledge no studies haveinvestigated the contribution of root-derived pectin for beneficialplant-microbial interactions.

Pectin degradation occurs through the action of many differentpectinolytic enzymes that are found in bacteria, fungi and higherplants. Protopectinases hydrolyze protopectin that exists in aninsoluble form within plant tissues, resulting in soluble pectin, andhave been observed in a wide range of Bacillus sp. (35). Many bacteriaare known to secrete pectin lyases to degrade plant pectin. This pathwaywas first reported in Escherichia coli (36) and pectinolytic activityhas been shown in the following bacterial genera: Achromobacter,Arthrobacter, Agrobacterium, Bacillus, Clostridium, Erwinia,Pseudomonas, and Xanthomonas (35). Many of these bacteria are recognizedas plant pathogens, and the degradation of pectin is a characteristic ofsoft rot disease as caused by Erwinia species. Among soft rot pathogensthere is evidence that pectinolytic activity is inducible and highlyexpressed compared to low-level constitutive expression of pectinolyticactivities by non-soft rot bacteria (37); therefore, the competition forpectin as a nutrient source within rhizospheres could be a mechanism bywhich beneficial PGPR strains (e.g., Bap) antagonize plant pathogenswithout themselves causing plant damage. We have experimental evidencethat our sequenced Bap strains encode and express a pectin lyaseactivity (FIG. 1).

By producing and secreting pectinolytic enzymes, bacteria can degradepectin and uptake the pectin-derived sugars D-glucuronate,D-galacturonate and D-mannose (38), which can be taken up by bacteriavia a hexuronate transporter (exuT). We found that the exuT gene isconserved among all sequenced PGPR Bap strains (7), and using anexuT-specific primer set we also found that this hexuronate transportergene was present in all of the Bap strains in the Auburn collection(data not shown). A similar approach confirmed the universal presencewithin these Bap strains of the uxuB gene, which encodes D-fructuronateoxidoreductase that is one of the enzymes responsible for utilizingpectin-derived sugars (39). Furthermore, we have experimental evidencethat our sequenced PGPR strains encode and express pectinolyticactivity, and can use the resulting monosaccharides as a sole carbonsource (data not shown).

Since the acquisition of carbon is essential for bacterial physiology wehypothesized that increased availability of pectin could promote thesurvival, persistence and metabolic activity of PGPR strains withinplant rhizospheres, leading to improved plant growth and diseasecontrol. There have been two previous reports on the use of exogenouslysupplied pectin to stimulate plant-associated bacteria. In the case ofnitrogen-fixing Azospirillum isolates, soils supplemented with pectinshowed an increase in Azospirillum growth (40). There is also evidencethat plant polysaccharides can induce biofilm formation in B. subtilisstrains (5). However, in previous studies there was no attempt toevaluate any benefit for plant growth. In our preliminary studies, wehave observed that there is a synergy between PGPR strains and soilamendment with pectin, resulting in statistically significant increasesin root and shoot weight (FIG. 3). The soybean root and shoot growthenhancement with both Bap and pectin amendment was observed with bothPGPR strains, with greater than a 2-fold increase in root weight (dry orwet) compared to the water control.

Interestingly, we also observed an over 10-fold increase in nodulation(FIG. 4B), with the size of the nodules produced in plants that had aPGPR inoculum and pectin supplementation greatly increased compared to aBap strain alone (FIG. 4A). Whereas we had predicted the increased rateof root and shoot growth, the finding that nodulation was enhanced was aserendipitous discovery. We hypothesize that increased pectinolyticactivity from the Bap plus pectin combination may result in enhancedBradyrhizobium infection of soybean roots, since it has been shownbefore in the model legume Lotus japonicus that a mutant lacking pectatelyase activity was also deficient in nodulation (41). Based on theincreased nodule frequency and size (FIG. 4A), we predict that therhizobia metabolic activity within the nodules is increased relative tothe no pectin controls, and that this results in greater nitrogenfixation rates. We further anticipate that the use of a pectin-rich soilamendment together with PGPR inoculants will be a cost-effective andsustainable method to promote plant growth and reduce the variabilityinherent in the use of beneficial PGPR strains in field soils.

Research Plan

(Objective 1) Identify & characterize novel plant rootcarbohvdrate-utilizing and pectin-utilizing microorganism. Surprisingly,a search of the literature for a microbial growth medium containing a“root extract” only yields a single publication in which soluble rootexudates were used to enrich for root-associated Archaea (42). Whilecitrus pectin-degrading microbes have been previously identified fromsoils (43, 44), to our knowledge no previous research effort has usedplant root-derived complex carbohydrates incorporated into a growthmedium to culture plant root-associated microorganisms. We will use aminimal medium containing either root extract or purified root pectin asa sole carbon and energy source, and will use long-term incubationconditions as have been done previously by our research group and othersto culture soil bacteria that have not been previously cultivated underlaboratory conditions (45, 46). In a preliminary study using soybeanroot incorporated into a minimal medium and inoculating agar plates withserially diluted soybean rhizosphere samples, we have isolated 184bacterial isolates that formed colonies after 3 months of incubation.Among these, we have completed sequencing the 16S rRNA gene from 144isolates, of which 39% have a 16S rRNA gene with a % identity <97% totheir top hit in the GenBank database! These bacterial isolates includerepresentatives from multiple bacterial phyla (i.e.,alpha-Proteobacteria, beta-Proteobacteria, gamma-Proteobacteria,Bacteroidetes, and other phyla) and genera (e.g., Inquilinus,Chitinophaga, Herbaspirillum, Flavobacterium, Delftia, Ralstonia,Burkholderia, and Dyella). Interestingly, of the 22 unique isolates thathave <97% identity to the top BLAST hit, 10 (45%) had top BLAST hits topreviously uncultured soil bacteria. In this proposed study we willculture from multiple soil types and use root homogenates and rootpectin to greatly expand our culture collection to identify microbialisolates with potential as PGPR strains. In a preliminary experiment, wehave observed that two of our previously sequenced PGPR Bap strains,AP143 and AP193, could utilize pectin as a C source for growth (FIG. 2).As a negative control, a non-PGPR Bacillus thuringiensis subsp. kurstakistrain HD73 was obtained from the USDA ARS culture collection (Ames,Iowa) that was identified as a non-pectin utilizing strain based on itsgenome sequence. This strain was not observed to grow using pectin as asole C source (FIG. 2).

(1A) Culture rhizosphere microorganisms: using a root extract medium.

Hypothesis: The use of plant root-derived complex carbohydrates as a Csource will enable cultivation of diverse endophytic and rhizospheremicroorganisms.

Experimental Methods: The bacterial and fungal assemblages associatedwith plant roots will be cultured using long-term incubation on aminimal medium containing either a plant root extract or purifiedpectin.

Root extract preparation: Root extract will be prepared using 10 corn(Zea mays) or soybean (Glycine max) roots grown in sand in pots in agreenhouse for 21 days. The washed roots will be homogenized with amortar and pestle after freezing under liquid nitrogen, and then thehomogenized roots will be suspended in sterile water in a 50 mlcentrifuge tube. After centrifugation, the supernatant containingsoluble root exudates (simple sugars and amino acids) will be removed.This washing will be repeated twice, and the root homogenate will bedried in a clean hood. The dry weight of the root homogenate will beused to prepare a suspension of 1 g root tissue per 10 ml sterile water.The root suspension will then be thoroughly homogenized using a T10Basic Ultra-Turrax dispersion unit (IKA Works, Inc., Wilmington, Del.)which is capable of generating sub-micron sized particulates suitable asa microbial growth substrate.

Pectin purification: Commercially available pectin is derived fromcitrus or apple sources, and is chemically different from root pectin(47). Therefore, we will extract pectin from corn or soybean roots,using approximately 50 corn or soybean plants grown at the same time asthe plants above. The washed roots will be shipped to the ComplexCarbohydrate Research Center (CCRC) where the root pectin will beextracted by using a 0.5% ammonium oxalate buffer containing 0.1% NaBH₄(pH 4) in a boiling water bath for 1 h each and pooling the supernatantsas described previously (48).

Root extract medium (REM) preparation: The root or pectin extract willbe added to a M9 salts minimal defined medium at a final concentrationof 4 mg per ml based on preliminary experiments with a range of rootextract concentrations (data not shown). After the addition of 1.5% agarand autoclaving the suspension, trace elements (FeCl₃, ZnCl₂, CuCl₂,CoCl₂, H₃BO₃, and MnCl₂) will be added to the REM according to therecommended concentrations (49) and plates will be thickly poured (30 mlper plate) after magnetic stirring. There will be a total of 4 differentmedia prepared (corn root, corn pectin, soybean root, soybean pectin)for cultivation efforts.

Cultivation of microorganisms: Soils will be sampled from activelygrowing corn or soybean plants in distinct soil types (clay, sandy andloam soils) within proximity to Auburn University. The sandy soil willbe collected from the Cullars Rotation in plots that have not received Kor P inputs for over 100 years, but yet have diverse microbialcommunities (50). Intact roots with associated rhizosphere will besampled from five plants per site and then pooled together. In order toaccess both endophytic and rhizosphere microorganisms, sub-samples willbe suspended in sterile water (10% w/v) and homogenized to produce afine suspension of microbial cells. This microbial suspension will beserially diluted, and the 10⁻⁴, 10⁻⁵ and 10⁻⁶ dilutions will be platedonto the four REM medium types with 10× replication (a total of 30plates per media type per soil site; 120 total plates per soil site).From previous experience, the 10⁻⁵ and 10⁻⁶ dilutions provide isolatedcolonies on a soybean REM whereas the 10⁻⁴ dilution tends to beovergrown with microbial colonies. The inoculated plates will be wrappedin parafilm and maintained in containers in which humidity is keptelevated to avoid plate desiccation. The plates will be incubated for2-3 months with regular monitoring for the appearance of microbialcolonies. After incubation, colonies will be passaged onto the same REMtype as their original isolation to obtain isolated colonies from purecultures. Pure cultures will be documented for their colony type bytaking digital photos using a digital microscope and will then becryopreserved in REM broth with 20% glycerol, prior to storing replicatetubes at −80° C. The primary isolation of plant rootcarbohydrate-utilizing microorganisms will be repeated as necessary toachieve several thousand pure microbial cultures in our collection.

Expected results: A large culture collection of plant-associatedmicroorganisms that can utilize plant root-derived carbohydrates will beobtained that include many novel microbial taxa.

Anticipated problems and their solutions: Based on our previous effortsto cultivate previously uncultured bacterial taxa from soils (46), aprinciple challenge is maintaining the viability of laboratory cultures.Sequential passaging may lead to loss of some cultures due toinsufficient nutrient availability or other unknown factors. Therefore,each culture will be cryopreserved at passage one when a pure culturehas been obtained so that if a culture losses viability that it may beresuscitated on a different growth medium, especially if there ispromising data generated regarding phylogenetic affiliation orbeneficial plant interactions. It is also anticipated to be difficult tocontinually prepare root extract medium agar plates because thisrequires growing corn and soybean plants for medium preparation. We willtherefore explore the use of commercially available root extracts thatare known to be pectin-rich (e.g., beet pulp) to prepare REM agar andbroth in order to maintain laboratory cultures for subsequentexperiments.

(IB) Conduct a phylogenetic analysis of cultured microbial isolates.Hypothesis: The bacterial and fungal isolates obtained from Obj. 1A willinclude recognized plant pathogens, beneficial microbes as well as novelcultured isolates that have not been previously been characterized fortheir plant interactions.

Experimental Methods: Each of the pure microbial cultures obtained inObj. lA will be subjected to a molecular phylogenetic analysis based ona comparison with 16S rRNA sequences for Bacteria (or Archaea, shouldthese be isolated) and internal transcribed spacer (ITS) analysis forfungal isolates. A portion of the pure culture from passage one will beswabbed from the REM agar plate into 1 ml of sterile water within amicrocentrifuge tube, and after centrifugation at 10,000×g the microbialpellet will be used to isolate genomic DNA using an E.Z.N.A. genomic DNAisolation kit (Omega Biotek, Atlanta, Ga.). The genomic DNA will be usedas a template for a PCR in which ‘universal’ bacterial 16S-specific (27Fand 1492R (51)) or fungal ITS-specific (ITS86F and ITS4 (52, 53)) primersets are used in order to generate an amplicon from each isolate. ThePCR product will be purified over a column and used for Sangersequencing with a forward or reverse primer (e.g., 27F and 907R)sufficient to provide a consensus sequence over a large portion of theamplicon. The consensus 16S rRNA gene or ITS sequence will be generatedusing the CLC Genomics Workbench (Qiagen, Cambridge, Mass.) and thesewill be subjected to a multiple sequence alignment and maximumlikelihood analysis (1000 iterations using RAxML (54)). Each of theunique isolate sequences will be compared against the databases atGenBank (nr/nt) and Ribosomal Database Project (RDP) in order toidentify the nearest neighbor for each of the microbial taxa. A recordof the % identity, nearest neighbor, and its phylogenetic affiliationwill be recorded for each isolate within a lab PlantMicrobiomeNetdatabase (see Data Management Plan), and if the isolate corresponds to aknown pathogen (e.g., Erwinia sp.) or beneficial microbe (B. subtilis)this will also be indicated within the database. Of particular interestwill be isolates that do not fall into either of the latter categoriesand have a relatively low % identity (<97%) to known culturedmicroorganisms within GenBank or RDP. Based on these results we willassemble 96-well plates containing a glycerol stock of each of theunique isolates, in triplicate, that is not affiliated with a knownpathogen. We will group the isolates based on their relative rate ofgrowth so that each 96-well plate contains isolates that will takeapproximately the same amount of time to reach the stationary phase ofgrowth.

Expected results: The phylogenetic affiliation for each of the purecultures obtained in Obj. 1A. Other outputs from this objective will bea collection of genomic DNAs from each isolate, an indication of whethereach culture represents a taxon that has not been previously cultured invitro or been associated with plants, and a 96-well plate formattedculture collection.

Anticipated problems and their solutions: This objective has a highprobability of success. If an isolate does not provide a PCR amplicon,which could be due to the primer set not amplifying from uniquemicrobial taxa, alternative primer sets will be used; if necessary,genomic DNA will be further purified using a method developed by the PI(55).

(1C) Screen cultures far plant hormone synthesis: and the ability topromote plant growth. Hypothesis: Many of the microbial isolates willhave the ability to induce plant hormone expression and will be able tostimulate plant growth.

Experimental Methods: Each of the pure cultures that have beenidentified as non-pathogenic in Obj. 1B will be used to inoculate A.thaliana that express green fluorescent protein (GFP) in response toplant hormones, and to test for soybean growth promotion.

Broth culture of microorganisms: Each 96-well cryopreserved platecontaining the unique, non-pathogenic microbial cultures (bacterial andfungal) in triplicate will be used to inoculate a 96-well, deep-wellplate with each well containing 1 ml of REM broth. Each plate will alsocontain controls inoculated in triplicate wells, the positive controlwill be B. amyloliquefaciens FZB42 which has been previously shown toproduce auxin (56), and the negative control will be Bacillusthuringiensis HD73 that does not produce any plant hormone. Abreathable, sterile film will be placed over each 96-well plate whichwill be shaken at 200 rpm for approximately 1 week. The exact length oftime will depend on preliminary experiments to establish the rate ofgrowth for representative isolates in this REM broth medium. Each platewill be monitored for the growth of the cultures and used when thecultures have attained sufficient turbidity (OD₆₀₀>0.5) to inoculatewells containing the A. thaliana bioassay.

Bioassay for plant auxin or cytokinin response: Auxin and cytokinin areessential plant hormones involved in root growth and are likely to beinvolved in the promotion of plant growth by PGPR strains. Since directmeasurements of these plant hormones is both difficult and timeconsuming, indirect fluorescent reporter lines have been generated andare standardly used to analyze auxin (DR5) and cytokinin (pTCSn) (57,58). We have bred each reporter line linked to a different fluorescenceprotein together to generate a single plant wherein both auxin andcytokinin response can be examined simultaneously. This hormoneresponsive line contains both DR5-YFP and pTCSn-GFP reporters. Seeds ofthis hormone-responsive line will be added to 96-well plates in which0.5 ml of molten (42° C.) 1.4% agar in water has been added to eachwell, and then a multichannel pipettor will be used to transfer 0.5 mlof the respective microbial culture to each well and will be mixed bypipetting. The seedlings will be grown for five days under standardgrowth conditions and then roots will be analyzed for changes inresponse to auxin and cytokinin levels based on altered fluorescenceusing an epifluorescence microscope. Both positive (hormone treated) andnegative (blank) controls will be examined in parallel and replicated todetermine the results for this objective. Positive results of hormoneresponse will be confirmed using qPCR expression analysis of routinelyexamine auxin and cytokinin responsive genes (e.g. IAAs and type-ARRs)as described previously (59).

Greenhouse bioassay for plant growth promotion: The microbial isolateswill also be tested for their respective ability to promote soybeanplant growth in an Alabama field soil in a greenhouse. The experimentswill be designed in a randomized complete block design in triplicate.Conetainers that are 2.5 inches in diameter and 8.25 inches deep will befilled with the same weight of thoroughly mixed field soil. Prior toplanting, the soil water content in conetainers will be adjusted to 60%field capacity. At planting, one soybean seed will be placed into thecenter of each conetainer at a depth of 1 inch, followed by pipetting0.5 ml of the respective microbial suspension over the top of each seed,then covering the soybean seed with soil. Watering conetainers willbegin 24 hr after planting. PGPR Bap strain AP193 used as a positivecontrol; B. thuringiensis strain HD73 and water only will be used asnegative controls. Plants will be transferred to the greenhouse andwatered daily for 21 days, after which the dry shoot and root weightswill be determined. For each isolate's impact on plant root and shootgrowth the variation will be compared to controls and analyzed usingANOVA at 5% level of significance.

Expected results: Identification of specific microbial cultures thatproduce plant hormones and/or induce plant growth that could be used asPGPR strains.

Anticipated problems and their solutions: There is inherent variabilityin bioassays that may result in false negative or positive results. Byconducting both laboratory (A. thaliana) and greenhouse assays in thisobjective we will increase the likelihood of observing biologicallymeaningful plant growth stimulation. The isolates that promote plantgrowth and/or produce plant hormones will be further tested in a potatoslice assay (60) to eliminate any isolates with the potential forinducing soft rot. We will select specific strains based on theirpositive effects on plant growth to advance to field studies to assesstheir potential for plant growth promotion and/or disease biocontrol,and these studies will be conducted using other funding sources.

(Objective 2) Evaluate the ability of exogenous pectin supplemention toenhance PGPR-mediated plant growth.

In preliminary experiments we have observed that amending soil withcitrus-derived pectin can significantly enhance PGPR-mediated plantgrowth (FIG. 3) and nodulation frequency and size (FIG. 4). In thisobjective we will investigate the effect of pectin concentration on PGPRstrain growth in soils; furthermore, we will assess the effects of soiltype and pectin source on PGPR-mediated growth enhancement of corn andsoybean plants. Lastly, we will determine the impact of A. thalianamutants that modulate root pectin levels on PGPR-mediated growthpromotion. These studies will advance the agricultural applications ofthis science and foster our understanding of the role of pectin inmediating plant-microbial interactions.

(2A) Determine pectin dose-dependent enhancement of PGPR strain growth.

Hypothesis: There will be a pectin dose-dependent increase in Bap growthwithin the rhizosphere.

It is important to first determine the concentration of pectin to use insubsequent plant growth experiments. Our initial experiments evaluateddifferent pectin (citrus source) concentrations from 0.001% to 5% onsoybean seed germination, and observed slightly inhibited germinationrates at concentrations >1%, and our initial greenhouse trials havetherefore been conducted using 0.1% pectin amended as a dry ingredientto soils.

Experimental Methods: In order to track strain colonization in soils wehave identified rifampicin (Rif)-resistant mutants using 50□ g/ml Riffor Bap strains AP143 and AP193, and these Rif^(R) mutants have similargrowth and PGPR activity compared to the wild-type strains (data notshown). A negative control strain, B. thuringiensis strain HD73, whichdoes not use pectin as a carbon source (61) and does not have PGPRactivity (data not shown), was also used to select a Rif^(R) mutant. Theexperimental system for assessing root colonization by the Rif^(R)mutants will be the same greenhouse tests as described above in Obj. 1Cusing soybean seeds grown in field soil. Seeds will be inoculated at thetime of planting with 1.0 ml of AP143, AP193 or HD73 Rif^(R) spores (10⁶colony forming units (CFU)/seedling) applied in soils that receive 1) noamendment, 2) 0.01% (w/w) pectin 3) 0.025% pectin, 4) 0.05% pectin, or5) 0.1% pectin, with 8 replications for each treatment group. At 21 dayspost-inoculation, 10 g of rhizosphere soil will be sampled from each ofthe pots, and the dry shoot and root weights will be determined. Thenumbers of nodules formed per soybean plant will also be determinedalong with the average nodule mass. The soil will be homogenized in 90ml of sterile water (10⁻¹ dilution) and then serially diluted to 10⁻⁶dilution and each of the dilutions from 10⁻¹ to 10⁻⁶ will be plated ontoTSA plates containing 50 □ g/ml Rif in order to determine the number ofRif^(R) CFU/g of soil for each of the PGPR strains and the negativecontrol strain.

Expected results: Determination of the pectin dose-dependent increase ingrowth of the PGPR Bap strains when amended into soil. The results ofthis experiment are expected to show a significant pectin dose-dependentincrease in the CFU/g of Bap strains AP143 and AP193, with nosignificant increase observed for pectin-incompetent strain HD73. Theseresults will enable the selection of a pectin concentration to use inObj. 2B. We will also evaluate the degree of nodulation enhancement andfuture studies will investigate the molecular basis for the increase innodulation frequency and metabolic activity associated with pectin andPGPR inoculation.

Anticipated problems and their solutions: To confirm that the CFU countsof the Rif^(R) are due to the inoculated PGPR Bap strains,representative colonies will be selected to conduct PCR using an AP143or AP193 strain-specific PCR primer set that has been developed in theLiles lab (data not shown). Our preliminary experiments have not shownany Rif^(R) colonies identified from plants that have not beeninoculated with a Bacillus Rif^(R) strain.

(2) Evaluate pectin enhancement of PGPR-mediated plant growth promotionusing multiple pectin sources, plants and soils. Hypotheses: There willbe a significant increase in plant growth in response to PGPR and pectinamendment compared to PGPR inoculation alone. Soil amendment with beetpulp will provide similar results in enhancing PGPR-mediated plantgrowth as with citrus-derived pectin.

Experimental Methods: We will conduct this greenhouse experiment inseparate trials using soybean and corn plants. In each separate trial,plants will be will be sown in trays that contain a sandy loam soil(Cullars Rotation soil) or a field soil used routinely in greenhousestudies, and 3 weeks later transplanted into 8 inch pots that containthe respective soil. As with previous experiments we will incorporatepectin into soil as a dry ingredient, and we will use a concentrationdepending upon the results of Obj. 2A. The PGPR strain will be appliedas a 1 ml drench to each seedling three days after transplantation. Inaddition to the commercial source of citrus pectin, we will alsoevaluate the use of powdered beet pulp as an alternative andcost-effective, pectin-rich amendment (62). The treatment groups willinclude 1) no PGPR in loamy soil, 2) no PGPR in clay soil, 3) 10⁶CFU/seedling PGPR Bap strain AP193 in loamy soil and 4) 10⁶ CFU/seedlingPGPR Bap strain AP193 in clay soil. These four treatment groups willeach be incubated with soil without pectin, with citrus pectin or beetpulp (n=12 treatment groups with 8 replicates=96 pots per eachexperiment). A completely randomized blocked design will be used. Freshand dry weight of root and shoot will be measured at the completion ofthe greenhouse experiment after 4 weeks. Root morphology will beanalyzed by a WinRHIZO root scanner (Regent Instruments, Quebec City,Canada). The numbers of nodules formed per soybean plant will also bedetermined along with the average nodule mass. For each parameter thevariation of each treatment will be compared and analyzed using ANOVA at5% level of significance (SAS 9.1 software). These experiments will berepeated with soybean and corn, and if additional low-cost sources ofpectin are identified we will evaluate the use of these alternative soilamendments to enhance PGPR-mediated plant growth promotion.

Expected results: The addition of citrus pectin or beet pulp toBap-treated soybean seeds will result in an increase in soybean root andshoot growth relative to non-pectin-treated plants. No pectin-mediatedincrease will be observed in the plants without a PGPR strain inoculum.

Anticipated problems and their solutions: There can be unforeseenissues, such as a disease outbreak, in experimental plants. We will takeample precautions to limit disease incidence, and plan to repeat thisexperiment at least once and more if necessary. The timing of theexperiment may need to be extended to achieve significant differencesamong treatment groups.

(2C) Evaluate PGPR colonization and persistence in wild-type Arabidopsisvs. pectin biosynthesis mutants. Hypothesis: A reduced PGPR-mediatedincrease in plant growth performance will be identified in Arabidopsisthat have a defect in pectin biosynthesis relative to wild-type.

Because A. thaliana does not exhibit a distinct root border cellseparation as with other plant species (63), it will be of interest toevaluate PGPR-dependent growth responses for wild-type and mutant A.thaliana.

Experimental Methods: As discussed above pectin is a complexpolysaccharide for which there are many biosynthesis and catabolismpathway steps. Because of this there are numerous genes involved inthese processes, and subsequently in Arabidopsis there are numerouspotential mutants to be examined that could have altered pectin levels.However, even in the well-studied Arabidopsis system only a handful ofgene mutants have been shown to have altered pectin levels in roottissues. We will examine the two best characterized mutants with defectsin pectin biosynthesis that are root expressed, quasimodo1 and 2 (qua1,qua2). The QUA1 gene encodes a glycosyltransferase (also known as GUAT8)and its mutant qua1 has been shown to have 25% less homogalacturonan(HG) and have a reduction in pectin esterification (64). QUA2 is apectin methyltransferase and its mutant qua2 has 50% less HG with normalesterification levels (65). Importantly, both mutants in these genesshow alterations in root border cell separation (reduced cell adhesion)resulting in distinct border cell separation typically observed in theother crop species (soybean and corn) examined in this proposal (63). Toour knowledge these are the only cell adhesion mutants that have pectindefects in roots. The selected qua1 and qua2 mutants have a dwarfedstature resulting from their reduction in pectin biosynthesis, althoughthese mutants are still viable (8, 65, 66). While there are a few otherArabidopsis mutants in pectin-related biosynthesis genes that areexpressed in root tissues, these either show no change in growthphenotype possibly due to the large degree of redundancy in this pathwayor have little information regarding changes in pectin levels (67). TheBap strain AP193 will be inoculated onto wild-type, QUA1, or QUA2 mutantA. thaliana seeds at an inoculum of 10⁶ CFU per seed such that theimpact of mutant phenotype on PGPR-mediated growth response can beassessed and compared to negative controls (water or B. thuringiensisstrain HD73). Inoculated seeds will be germinated and grown underseveral standard conditions (96-well plate as noted above, standard agarplates and soil growth chamber conditions). Plants will have basicgrowth (leaf size and number, silique and seed production) and health(Fv/Fm chlorophyll fluorescence) measured at distinct stages acrossdevelopment to determine PGPR-mediated effects. Measurements at allstages will be performed with a minimum of 10 plants per treatment groupand measurements will be analyzed as noted above.

Expected results: Reduced PGPR-mediated growth will be observed in theA. thaliana pectin synthesis mutants compared to the wild-type control.

Anticipated problems and their solutions: Some alleles of the qua1 andqua2 selected mutants have extreme dwarf growth phenotypes that wouldmake the proposed examinations difficult to conduct. As such we willselect more robust alleles that still have pectin defects, readilyavailable from the Arabidopsis stock center to conduct theseassessments.

(Objective 3) Determine the identity and functions associated with rootpectin-metabolizing soil microorganisms using culture independentapproaches.

The experiments described above rely upon culture-dependent studies ofnovel rhizosphere microorganisms and known PGPR strains. While the useof cultured PGPR strains has great utility for application inagriculture, we also know that the vast majority of soil microorganismsare not readily cultured under laboratory conditions (68). In thisobjective we will therefore use stable isotope probing (SIP) with¹³C-root pectin in order to study the phylogenetic diversity andfunctional contributions of soil microorganisms that utilize root pectinas a C source. We will complement this culture-independent approach bystudying microbial genomes recovered from a soil metagenomic librarythat are predicted to encode pectin-degrading or -utilizing functions.

(3A) Determine the phvlogenv and functions of soil microbial taxa thatutilize ¹³C-pectin. Hypothesis₁: The pectin-utilizing microorganismsidentified will include those identified in Objective 1 and will alsoinclude other microbes that have not been previously associated withinpectin utilization or characterized as plant-associated. Hypothesis₂:The pectin-utilizing microbes will express gene products that arepredicted to be important for plant growth.

Experimental Methods: ¹³C-Stable Isotope Probing. We will evaluate thesoil microbial use of ¹³C-root pectin with and without the presence ofthe PGPR Bap strain AP193. The Cullars Rotation soil (described above)will be added to trays in which corn seeds will be sown. After threeweeks the seedlings will be transplanted into 4.5 inch pots containingsoil with and without 0.1% root pectin (w/w) extracted from corn rootsby the CCRC (see above). Additional pots will be planted to guardagainst loss of plants from disease and to serve as controls and formethod refinement. For the appropriate treatment groups, three daysafter transplantation a 10 ml PGPR inoculum will be applied as a 10 mlroot drench (10⁶ CFU/seedling) and then all plants will be grown for 3weeks in a greenhouse prior to addition of the ¹³C-pectin inoculation.This is comparable to a study in which ¹²C-carbohydrate amendments (butnot pectin) were added to soils for 3 weeks prior to addition of the ¹³Clabel (69). As in this study, we will assess CO₂ production fromreplicate pots receiving unlabeled root pectin at differentconcentrations using headspace-collected CO₂ and will measure CO₂ at theAuburn University Mass Spec facility using a Shimadzu GC-2014 gaschromatograph, using multiple time points post-pectin addition in orderto establish the appropriate concentration of ¹³C-pectin to add and thetime frame for sampling. Our ability to identify the soil microorganismsthat are responsible for actively degrading and incorporating ¹³C-pectinwill be enabled by isolating RNA from soils which is more labile andrepresentative of metabolically active microorganisms. Unlike readilymetabolized substrates such as ¹³C-glucose in which RNA-SIP is conductedwithin 12 hours (reference), a more complex polysaccharide such aspectin should require a multiple day time frame for this study. Therewill be two treatment groups (control, PGPR)×4 replicates×4 time pointsfor a total of 32 pots. The ¹³C-labeled root pectin will be prepared bypurchase of 20 g uniformly ¹³C-labeled [97 atom % ¹³C] corn root(IsoLife, Wageningen, The Netherlands), and pectin will be extractedfrom root tissue by the CCRC as described above at the scale needed tohave sufficient root-derived pectin for soil amendment (estimated yield˜4 g ¹³C pectin). Each pot will contain ˜100g of soil, therefore ourtotal needed yield of ¹³C-pectin will be 0.1 g per pot ×32 pots=3.2 g.Four time points will be sampled, at time zero just prior to a ¹³C-rootpectin inoculum equivalent to the concentration used in previousexperiments (0.1% (w/w)) in a 10 ml volume and then at threepost-inoculation time points (˜24, 48 and 72 hours, depending on theresults of CO₂ production). At each time point we will sample therhizosphere soil from each plant, adding approximately 2 g ofrhizosphere soil to 5 ml of LifeGuard™ Soil Preservation Solution(MoBio, Carlsbad, Calif.) in a 15 ml collection tube, and a replicatetube will be frozen at −20° C. which will remain viable for RNA/DNAisolation for>30 days. Total DNA and RNA will be isolated from 2 g ofsoil sample using the PowerSoil Total RNA Isolation kit (MoBio) in adedicated RNA processing area of the PI' s lab. DNA will be separatelyeluted using a DNA Elution Accessory kit (MoBio) and RNA and DNA sampleswill be quantified using a Qubit fluorometer. The RNA samples will beseparated on a cesium trifluoroacetate isopycnic gradient subjected toultracentrifugation at 128,000×g for 50 hr using a Beckmanultracentrifuge. Control gradients using a pure bacterial (Bap AP193)culture grown on ¹²C-pectin or ¹³C-pectin will be conducted in advanceto establish conditions that result in good separation of light fromheavy RNA (70), and a syringe pump will be used in order to provide alow flow rate (˜200 □1/min) for fraction collection in sterileRNAse-free microcentrifuge tubes. We will quantify the amount of RNA ineach 100 □1 fraction using a rapid qPCR method targeting bacterial rRNA(71) using SYBR Green fluorescence in a Bio-Rad CFX real-time PCR system(Hercules, Calif.) in order to establish migration of light and heavyRNA. Fractions with abundant extracted RNA will be pooled for thefractions exhibiting clear separation of heavy from light RNA, treatedwith DNase I, and then reverse transcribed using M-MLV ReverseTranscriptase (Invitrogen, Carlsbad, Calif.). The resulting cDNA will beused for shotgun metagenomic sequencing.

Microbiome analysis: The ¹³C-labeled cDNA and total DNA recovered fromeach sample (32 samples, with cDNA and DNA, for a total of 64 samples)will be used as a template for 16S rRNA and gyrB gene PCR amplification.While the 16S rRNA gene is the most common molecular target inmicrobiome studies, it produces a distorted representation of microbialrelative abundance due to differing copy numbers of the rRNA operonwithin microbial genomes. We will use a two stage polymerase-exonuclease(PEX) PCR method in development with our collaborator Dr. Stefan Green(Univ of Illinois—Chicago) that results in improved evenness ofamplification across mixed templates and allows for greater primerdegeneracy (72). Using the cDNA or DNA templates, an analysis based onthe standard Illumina protocol for 16S rRNA gene sequence amplicons withthe 2-stage PEX-PCR protocol will be conducted for both 16S rRNA andgyrB sequences. Since gyrB is a housekeeping gene that is single-copy,we expect that this will result in a less biased representation of themicrobiome, and we have already generated a novel set of ‘universalbacteria’ gyrB primers that will be used in this study (data not shown).Using the QIIME pipeline and custom scripts, the 16S and gyrB sequenceswill be trimmed, binned into operational taxonomic units (OTUs) basedon >97% identity, and compared to existing phylogenetic databases todetermine the relative abundance as compared to the known colony formingunits. By conducting principal coordinate analysis from the 16S and gyrBsequence data we will reveal trends in the comparison of treatmentgroups at the different phylogenetic resolutions afforded through 16Sand gyrB, respectively. The known 16S rRNA and gyrB sequences for Bapstrain AP193 will be used for comparison.

Next-generation sequencing (NGS) and bioinformatics analyses: In orderto reduce the number of samples used for NGS analysis, we will evaluatethe concentration of heavy cDNA to identify samples in which there wasless recovered ¹³C labeled cDNA. Of the 32 total samples processed, weexpect to be able to eliminate one of the time points resulting in ˜24¹³C-cDNA samples. For comparison purposes we will also include the totalDNA sample recovered from each sample and each of the samples will bebar-coded for Illumina sequencing using the Nextera kit (Illumina, SanDiego, Calif.) using a unique index. We will include 8 samples perIllumina HiSeq lane in order to provide sufficient coverage per samplein order to better access lower abundant transcripts. The raw sequencereads will be trimmed for sequence quality equivalent to a Q score>30and the trimmed reads will then be used for de novo assembly using theSPAdes assembler running on the Auburn University supercomputer. We willcompare the transcriptome from each sample and treatment group using theCLC Genomics Workbench in order to identify differentially expressedtranscripts in each treatment group and to determine the highlyexpressed transcripts present within the ¹³C-enriched sample. We willsubmit the transcripts to the MG-RAST automated pipeline for microbialmetagenomic analysis and will in particular be seeking information onplant-related functions. Our specific molecular targets will includetranscripts related to nutrient (NPK) acquisition, plant hormoneproduction, and secondary metabolite synthesis, among other potentialfunctions. The contigs from each transcriptome will also be submitted tothe antiSMASH pipeline for biosynthetic cluster identification toprovide enhanced detection of biosynthetic clusters, with comparisons ofthe % identity and synteny of each discovered pathway with knownpathways from the GenBank database. These analyses will lead to testablehypotheses regarding pectin-utilizing microorganisms and theircontributions to plant growth.

Expected results: A culture-independent analysis of the pectin-utilizingmicroorganisms within the corn rhizosphere. This will indicate thephylogenetic affiliation and the expressed functions of thepectin-utilizing microorganisms, with a particular emphasis onplant-related phenotypes.

Anticipated problems and their solutions: One potential problem will bethat using a living corn plant will result in dilution of the ¹³Cenrichment for pectin-utilizing microorganisms. It will therefore benecessary to carefully monitor the degree to which providing exogenouspectin stimulates microbial respiration by the series of control potsusing non-labeled pectin prior to the ¹³C-pectin experiments. In thisway we can maximize the degree to which the RNA of the pectin-utilizingmicrobes has incorporate the ¹³C label and can be separated fromnon-labeled RNA.

(3B) Mine a soil metagenomie library for pectinolytic enzymes andassociated functions.

We have previously constructed a large-insert soil metagenomic libraryfrom the same plot of the Cullars Rotation soil that will be used forthe SIP study. High molecular weight DNA isolated from the soilmicrobiota (73) was randomly sheared and ligated into a bacterialartificial chromosome (BAC) vector, resulting in a metagenomic clonelibrary containing 110 kb average insert sizes with 19,200 clones. Thelibrary consists of 50 384-well plates, and based on the average insertsize the total amount of cloned metagenomic DNA is estimated to exceed 2Gbp, or over 500 Escherichia coli genome equivalents (data not shown). Anext generation sequencing strategy was used in which clones were pooledfrom each of the 50 plates, 24 columns and 16 rows separately and eachpool was uniquely bar-coded and sequenced using multiple Illumina HiSeqruns to achieve greater than 100× x average coverage per each of the19,200 clones. In other words, each of these metagenomic clones istheoretically represented within 3 different pools (column, plate androw) and by bioinformatically comparing these different sequencedlibrary pools we can identify the gene content and exact library welllocation for each clone (manuscript in preparation). The pooledsequences were processed to achieve high quality sequence reads andgenerate assembled, contiguous genomic fragments (contigs). In apreliminary search for microbial genomic regions involved inpectin-related functions, we searched for gene sequences related to thedegradation, uptake or utilization of pectin and pectin (orpectate)-derived sugars. Interestingly, we discovered 75 uniquemetagenomic contigs that had significant (E<10⁻⁵) homology with aBacteroides pectate lyase gene, and when each of these gene homologswere queried against the GenBank database these gave a mean % amino acididentity of 62%, and only one metagenome-derived gene had a % identitygreater than 90%. Several hundred unique contigs were also identifiedthat have homology with pectin-derived sugar uptake or utilization genes(data not shown). Collectively, this indicates that there is a wealth ofpreviously undiscovered diversity of pectin-associated functions amongsoil microorganisms.

Sequencing of the BAC clones containing pectin-related functions: Eachof the BAC clones identified by querying using known pectin-related genesequences (including each gene known to be required for pectindegradation, uptake and utilization) will be identified for its exactlocation within the soil metagenomic library from a local BLAST searchagainst each of the respective pool of clones (plate, column and row).To validate that the correct clone has been identified, the respectiveE. coli BAC clone will be grown for isolated colonies onto LB mediumcontaining 12.5 □g per ml chloramphenicol and then a clone-specificprimer set will be designed and used to PCR amplify a small geneticregion unique to each clone (<500 bp). Each validated BAC clone willthen be bar-coded using a Nextera kit and included within a HiSeq run inorder to generate a complete insert sequence for each clone. The cloneinsert will be generated by trimming the sequence reads and de novoassembled using the CLC Genomics Workbench, and then the clone contigswill be submitted to the RAST automated pipeline for annotation. We willalso manually annotate clones to examine the predicted functions andrelated genes present on each genomic region. We will conductcomparative genomic analyses to examine pathways related to pectindegradation, uptake and utilization from diverse soil microorganisms,deposit these sequences in GenBank for public access, and compare thesesequences to those obtained from the SIP results (from the same soil) inorder to link transcriptome data within a larger genomic context.Because the sequences obtained from the transcriptome analysis areexpected to be short contigs all less than ˜5 kb, the availability ofgenomic contigs >100 kb from the same soil will enable linking encodedfunctions from abundant and metabolically active soil microorganismsthat metabolize pectin.

Expected results: A culture-independent metagenomic analysis of thepectin-utilizing genes recovered from soil microorganisms. This willresult in a unique dataset of large genomic regions recovered frommicroorganisms from the same soil used for transcriptome analysis.

Anticipated problems and their solutions: Much of the research hasalready been completed in order to construct the soil metagenomiclibrary and to generate an exhaustive sequence database from column,plate and row pools. This objective is therefore very low risk becausewe already know that there are diverse pectinolytic functions encodedwithin the library that can be accessed, sequenced, and compared to thetranscriptome data generated in Objective 3A.

Broader Impacts

We expect that this research will provide: 1) New beneficial microbialcultures that can be used to promote plant growth in many agriculturalcrops. These cultures may have novel plant interactions and producemetabolites that promote plant growth and/or control plant pathogens. 2)The cost-effective application of pectin as a soil amendment in order toenhance microbial plant growth promotion. Field application of PGPRstrains is often ineffective, and we expect that the use of pectin willpromote better efficacy and result in improvement in PGPR-mediatedbiological effects. Our study will evaluate cost-effective pectin-richsoil amendments that could be used for agricultural application of thistechnology. 3) New insights into root pectin-utilizing microbes andtheir plant-related functions. Through the use of culture-independentapproaches we will provide information on the diversity ofpectin-utilizing microbes, the plant-related functions they express andprovide a database of their genomic regions associated with pectin use.4) We will provide educational opportunities as a component of thisresearch.

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Example 5 —Proposal: Enhancing Fish Nutrition, Growth Performance andDisease Resistance in Fish Using a Probiotic Bacillus and PrebioticPectin

Project Summary

Aquaculture sustainability is threatened by disease pressure and theeutrophic conditions resulted from intensive fish farming. Previousresearch conducted separately at Auburn University and at OceanUniversity-Qingdao has resulted in the identification of Bacillusamyloliquefaciens strains that have the ability to reduce mortality dueto multiple aquaculture pathogens. Studies in aquaria and ponds havedetermined that feeding fish with B. amyloliquefaciens probiotic-amendedfeed can enhance catfish growth performance resulting in a 8-14%increase in final weight as compared to control fish. Interestingly, inthe pond study there was also an observed improvement in water qualityin ponds in which fish were fed with the probiotic-amended feed, assignificant reductions were found in pond water total phosphorus (19%),total nitrogen (43%), and nitrate (75%), along with reduced levels ofchlorophyll and 2-methylisoborneol. Recent insights into the geneticsand physiology of the B. amyloliquefaciens probiotic strains indicatesthat these bacteria can use plant-derived pectin as a carbon and energysource. Because aquaculture feeds are plant-based, and these probioticbacteria were derived from plant rhizospheres, the use of plantcarbohydrates like pectin by the probiotic bacteria is expected toenhance probiotic-mediated effects such as feed conversion efficiencyand the production of secondary metabolites that antagonize aquaculturepathogens. This proposal will evaluate the hypothesis that thissynbiotic approach, the addition of a probiotic Bacillus together withthe prebiotic pectin to fish feed, will result in improved fish growthperformance and reduced mortality due to infection with an aquaculturepathogen compared to fish fed with a control diet or with the probioticor prebiotic alone.

Project Narrative and Justification

Due to their rapid growth rate, low cost, and proficient reproductioncapabilities, catfish species including the channel catfish (Ictaluruspunctatus) in the United States and the Southern catfish (Silurusmeridionalis Chen) in the People's Republic of China has become one ofthe more popular and economically important aquaculture species,particularly in the southeastern United States (USDA, 2003a; USDA2003b). For maximized productivity of the aquaculture system, fishfeeding efficiency is extremely important. Traditionally, foragefisheries have been exploited for generation of fish meal, but the rapiddepletion of wild fisheries (Naylor et al, 2009) has led to the use ofsoy meal as an alternative (Tacon, 1987; NRC, 2011). However, feedconversion ratios (FCR) are much lower in fish with the use of plantprotein resulting in up to a 15% deterioration of fish growthperformance (Sales, 2008). Due to the presence of 1-2% phytate contentin soy-based feed, over two-thirds of the phosphorous found in plantprotein sources are unusable by fish, and phytate can serve as ananti-nutrient in binding iron resulting in anemia (Zhu, 2014). Unusedphytate will ultimately be released as fish waste, contributing to theeutrophication of the aquaculture pond ecosystem (Lazzari, 2008).

Eutrophication due to indigestible components of the feed and excretednutrients can result in blooms of algae and cyanobacteria (Shaw et al,2003). Due to the ability of Cyanobacteria taxa to synthesize andrelease toxins into the water column, they can be devastating to fishproduction (Rodgers, 2008). In addition to hepatotoxins and neurotoxins,some Cyanobacteria and other bacterial taxa produce the metabolites2-methylisoborneol (MIB) and geosmin that result in off-flavors incatfish (Tucker and Ploeg, 1999).

Phytase is a phosphohydrolase that catalyses the hydrolysis of phytate,allowing for phosphorous availability for absorption (Kumar et al.,2012). This enzyme is found in many microorganisms, which are beingexploited for supplementation in feed. To supplement high feed demands,production facilities have been created to exponentially ferment phytasefrom microorganisms, many of which are already regarded as probiotics(Askelson et al, 2014). For this reason, providing the fish probioticsin their diet can potentially reduce eutrophication, induce weight gain,and be a viable option for preventing economic loss. Probiotic bacteriamay promote the growth of fish by improving feed nutrient quality and byremoving anti-nutrients such as phytate. Some microorganisms expressphytase activity, catalyzing phytate hydrolysis and allowing forphosphorous absorption (Kumar et al., 2011). Purified microbial phytaseshave been used as a feed additive in fish feeds to promote growth andreduce eutrophication (Kumar et al 2012)). For this reason, feeding fishwith a phytase-expressing probiotic could be a sustainable managementpractice to reduce eutrophication, induce weight gain, and result in analtered aquaculture pond ecosystem with reduced incidence of disease andoff-flavor.

Another factor responsible for significant economic losses inaquaculture is due to loss from disease (FDA, 2012). One traditionaltreatment for disease is the use of antibiotics, and there are currentlythree approved by the FDA for use in aquaculture production facilities(FDA, 2011). However, with growing concern over the use of antibioticsdue to the development of resistance in pathogens, it is important toseek alternative means of treatment. Probiotics can reduce mortality dueto pathogens by direct antagonism via synthesis of secondarymetabolites, by competitive exclusion, and by activation of the innateimmune system (Balcazar, 2006; Macfarlane, 1999; Wang, 2008). Bacillusspp. have good potential for aquaculture application due to theirability to form endospores, allowing for a long shelf life and survivalof gastric acid (Casula, 2002; Hong, 2005; Hyronimus, 2000).Furthermore, strains within the B. subtilis group, which includes B.amyloliquefaciens, have not been associated with disease.

Previous research at Auburn University by the PIs evaluated a collectionof 160 Bacillus spp. strains for their antimicrobial activity againstbacterial and fungal fish pathogens (Ran et al., 2012). The 21 Bacillusspp. strains that showed production of secondary metabolites thatinhibited the growth of Edwardsiella ictaluri, Aeromonas hydrophila andother pathogens were then tested for their survival and persistence inthe catfish intestine and protection against E. ictaluri infection (Ranet al., 2012). When Platydoras armatulus (striped catfish) were fed withspore-amended feed significant reductions in mortality relative to thecontrol group was observed after challenge with E. ictaluri (Ran et al.,2012).

The four best-performing Bacillus spp. strains (AB01, AP79, AP143 andAP193) were selected for further study for their potential for diseasecontrol and fish growth promotion. All four strains were found to beaffiliated with B. amyloliquefaciens based on phylogenetic analyses(Hossain et al., 2015), without any virulence-related geneticdeterminants (data not shown). Each of these B. amyloliquefaciensstrains was evaluated separately for their relative degree of channelcatfish growth promotion and biocontrol activity. Feed amended with B.amyloliquefaciens AP193 provided the greatest degree of fish growthpromotion in both replicated aquaria and pond studies, with a 8.5%(P<0.05) or 21.8% (P <0.1) increase in the average weight gain per fishcompared to fish fed with control feed in aquaria and pond studies,respectively. Furthermore, the fish fed with strain AP193 had amortality due to E. ictaluri infection of 47.8% compared to the 62.1%mortality rate for fish fed with control feed ((P<0.05). It waspreviously observed that strain AP193 expresses the antibioticdifficidin and that the production of this polyketide is critical forAP193 biocontrol activity in plants (Hossain et al., 2015). We haveobserved that strain AP193 mutants deficient in difficidin synthesis(□sfp or □dfnD) were also completely lacking in the ability to inhibitthe in vitro growth of bacterial fish pathogens such as E. ictaluri andA. hydrophila (data now shown), further supporting the hypothesis thatdifficidin production is important for fish disease control whileleaving open the possibility that other mechanisms (e.g. competitiveexclusion, stimulation of immune competence) are also involved.

Together with the increase in catfish growth performance and reductionin fish mortality, significant reductions in total phosphorus, totalnitrogen, and nitrate nitrogen levels were observed in ponds containingchannel catfish fed with AP193—indicating beneficial, pond-wide effectson water quality (Table 5).

TABLE 5 Mean concentrations mg/L of water quality parameters in controlponds and ponds fed with AP193 amended feed. Control AP 193 TreatmentWater Quality Parameter P-value (Mean ± SD) (Mean ± SD) Total Phosphorus0.014 0.136 ± 0.049a 0.110 ± 0.066b Total Nitrogen 0.025 0.344 ± 0.248a0.195 ± 0.120b Total Ammonia Nitrogen 0.829 0.142 ± 0.065a 0.137 ±0.059a Nitrite Nitrogen 0.945 0.004 ± 0.004a 0.004 ± 0.004a NitrateNitrogen 0.044 0.051 ± 0.095a 0.013 ± 0.026b

Excessive concentrations of N and P in ponds can contribute to densecyanobacterial or algal blooms that induce toxic eutrophication and fish“off-flavor” (Boyd, 2015). Soy-based fish feed contains high levels ofphytate, which is inositol-hexaphosphate (Cao, 2007; Storebakken, 1998).AP193 is known to encode a phytase (Hossain et al., 2015) and has beenobserved to express phytase activity (data not shown). Thus, AP193 hasthe capacity to degrade the phytate present within feed, resulting inmore iron availability to support fish growth as well as decreasingphosphate excreted from fish, thereby preventing the release ofphosphorus into the water that can result in eutrophication.Furthermore, previous research has determined that A. hydrophila has theability to use myo-inositol as a sole C source and suggests that thepresence of high levels of inositol in the diet could contribute to A.hydrophila pathogenesis (Hossain et al., 2013). By expressing a phytaseactivity, strain AP193 may be improving not only fish growth and waterquality, but also removing a key nutrient (inositol) that may contributeto A. hydrophila pathogenesis. This study will therefore investigate thebenefit of feeding fish with feed amended with AP193 in reducingmortality associated with virulent A. hydrophila that is known to causesevere losses to the aquaculture industries in China and the UnitedStates (Rasmussen-Ivey et al., 2016).

The probiotic effects observed to date support the potential benefit ofthis strategy for improving the sustainability of fish farming in Chinaand the United States. While promising, there is inherent variability inthe water quality and microbiology associated with aquaculture practicesin both countries, and there is a need to increase the efficacy ofprobiotics so that they can perform under various conditions. Ourprevious genomic study that investigated the predicted functionsassociated with these probiotic strains found that one trait that the B.amyloliquefaciens strains have in common is the ability to use the plantcarbohydrate pectin as a carbon and energy source (Hossain et al.,2015). We subsequently found that the ability to use pectin-derivedsugars was a universal characteristic of the 59 B. amyloliquefaciensstrains that had been used to promote the growth of plants (Adesemoye etal., 2009) and/or fish (Ran et al., 2012) in the Auburn collection. Thisled to the hypothesis that adding pectin to the fish diet could enhancethe growth of the probiotic bacteria and thereby improve fish growthperformance, water quality, and disease resistance. We have now testedthis hypothesis in soybean model together with Prof. Joseph Kloepper(Auburn University), and have observed a strong synergy between strainsand B. amyloliquefaciens pectin, resulting in statistically significantincreases in soybean root and shoot weight (data not shown). The soybeanroot enhancement using bacterial and pectin amendment was observed withmultiple B. amyloliquefaciens strains, including strain AP193, withsignificant root growth and nodulation enhancement observed when bothbacterial spores and pectin were added together compared to thebacterial treatment alone (FIGS. 3 and 4), and no effect on root growthwas observed when pectin was applied alone (data not shown).

Objective

This proposal will test the ability of pectin as a prebiotic to enhancethe probiotic effects we have previously established in catfish, whichis referred to as a “synbiotic” approach. Specifically, different dosagerates of pectin incorporated into the diet will be evaluated as to howprobiotics respond to this addition in the diet and by promoting fishsurvival after bacterial challenge with virulent Aeromonas hydrophila.This could directly benefit the sustainability and productivity ofaquaculture as practiced in both China and the United States.

Experimental design: Parallel studies will be carried out in the US andChina using Channel Catfish and the Southern catfish in conjunction withspecific strains of B. amyloliquefaciens. All trials will be designed todetermine the efficacy of select prebiotic and probiotic combinations ongrowth, nutrient retention and disease resistance under controlledconditions.

Aquaria Trials: At both institutes a commercial type basal plant-baseddiet will be formulated and produced in house using typical feedmanufacturing methods. The basal diet will then be modified by addinggraded level of pectin and suitable levels of the probiotic. The testdiets will then be offered to five replicate groups (15-20 fish/tank) ofjuvenile fish over a 10-week culture period. The fish will be offeredfeed twice daily at a level approximating satiation. The fish will bemaintained in a re-circulating culture system designed to maintainsuitable water quality parameters. Each system will consist of culturetank, UV sterilization of water, solids filtration, biologicalfiltration, circulation pump and supplemental aeration. At theconclusion of the growth trial survival, final weight, thermal growthcoefficient, percent weight gain and feed conversion efficiency will bedetermined. Additionally, a sub-sample of fish will be analyzed forproximate composition using standard AOAC procedures and the date usedto determine apparent net protein, phosphorus and iron retention.Meanwhile, the intestinal morphology will be analyzed, including thedimensions of both jejunum wall thickness and villus height in differentgroups.

TABLE 6 Aquaria Trials Channel Catfish (US) B. amyloliquefaciensSouthern Catfish (China) Treatment Diet type AP193 Diet type B.amyloliquefaciens 1 Basal diet (BD) Basal diet (BD) 2 BD + 0.05% BD +0.05% pectin pectin 3 BD + 0.10% BD + 0.10% pectin pectin 4 BD + 0.50%BD + 0.50% pectin pectin 5 Basal diet (BD) 10⁶ CFU/g Basal diet (BD) 10⁶CFU/g 6 BD + 0.05% 10⁶ CFU/g BD + 0.05% pectin 10⁶ CFU/g pectin 7 BD +0.10% 10⁶ CFU/g BD + 0.10% pectin 10⁶ CFU/g pectin 8 BD + 0.50% 10⁶CFU/g BD + 0.50% pectin 10⁶ CFU/g pectin

Disease challenge with virulent A. hydrophila ML09-119: All fish will betransferred to the disease challenge laboratory maintaining fish withineach replicate aquaria. Fish will be allowed to acclimate for one weekwhile being offered the same treatment diet as above. The fish will thenbe challenged by immersion with vAh using an immersion protocoldeveloped by the USDA-ARS in which fish have their adipose fin clippedand are then immersed in 10⁷ CFU/ml vAh for 1 hr, typically resulting in˜50% mortality within 24 hrs. The percent mortality in each treatmentgroup will be determined and confirmed with representative vAh casesusing a vAh-specific diagnostic tests. Statistical comparisons amongtreatment groups will be conducted with one-way ANOVAs with significanceassessed at P <0.05.

Timeline of Events: At about 0-5 months, the growth and diseaseexperimental trial will be performed. At about 4-9 months, tissue andhistological preparation and evaluation will be performed. At about10-12 months, data analysis and report preparation will be performed

Expected Results and Outcomes

As pectin levels are increased within diets containing the probiotic, weexpect higher growth rates and feed conversion efficiency, along withbetter survival after pathogen challenge as a result of increasedproliferation of the probiotic and increased production ofprobiotic-derived secondary metabolites (e.g. difficidin) within theintestines. We do not anticipate any affect on growth performance orsurvival upon bacterial challenge due to pectin being in diet alone.Additionally, we do not anticipate any adverse affects in the intestinallining due to the increased levels of beneficial bacteria, beingconsistent with previous studies conducted in our laboratories.

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It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention. Theinvention illustratively described herein suitably may be practiced inthe absence of any element or elements, limitation or limitations whichis not specifically disclosed herein. The terms and expressions whichhave been employed are used as terms of description and not oflimitation, and there is no intention in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention. Thus, itshould be understood that although the present invention has beenillustrated by specific embodiments and optional features, modificationand/or variation of the concepts herein disclosed may be resorted to bythose skilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention.

Citations to a number of patent and non-patent references are madeherein. The cited references are incorporated by reference herein intheir entireties. In the event that there is an inconsistency between adefinition of a term in the specification as compared to a definition ofthe term in a cited reference, the term should be interpreted based onthe definition in the specification.

We claim:
 1. An inoculant comprising: (a) Bacillus amyloliquefacienssubspecies plantarum (BAP), and (b) a saccharide comprising pectin or apectin-related saccharide.
 2. The inoculant of claim 1, wherein thesaccharide is a heteropolysaccharide comprising D-galacturonate monomerswhich represents at least 50% of all monomers of theheteropolysaccharide.
 3. The inoculant of claim 1, wherein thesaccharide is a heterogeneous mixture of polysaccharides ormonosaccharides comprising D-galacturonate monomers or D-glucuronatemonomers, and the sum of D-galacturonate monomers and D-glucuronatemonomers in the mixture represent at least 50% of the total monomers ofthe mixture.
 4. The inoculant of claim 1, wherein the saccharide ispectin having an average molecular weight of at least about 30000 g/mol.5. The inoculant of claim 1, further comprising a nitrogen-fixingbacteria.
 6. Plant seeds coated with the inoculant of claim
 1. 7. Theplant seeds of claim 6, wherein the seeds comprise at least two layers,the two layers comprising at least: (i) a first layer comprising theBacillus amyloliquefaciens subspecies plantarum (BAP); and at least (ii)a second layer comprising the pectin or the pectin-related saccharide,wherein the second layer is interior relative to the first layer. 8.Animal feed comprising the inoculant of claim
 1. 9. The animal feed ofclaim 8, wherein the feed comprises 10⁴-10⁷ CFU of the BAP per gram. 10.The animal feed of claim 1, wherein the feed comprises pectin at aconcentration (w/w) of at least about 0.05%.
 11. The animal feed ofclaim 8 formulated for farmed fish.
 12. The animal feed of claim 8,wherein the animal feed is coated with the inoculant.
 13. The animalfeed of claim 8, wherein the animal feed is prepared by forming amixture of the animal feed and the inoculant and forming a compressed orpelleted animal feed from the mixture.
 14. A method for increasingnodulation in a legume, the method comprising: (a) treating the legume,seeds of the legume, or soil surrounding the legume with Bacillusamyloliquefaciens subspecies plantarum (BAP) and (b) treating thelegume, the seeds of the legume, or the soil surrounding the legume witha saccharide comprising pectin or a pectin-related saccharide.
 15. Themethod of claim 14, wherein the legume, the seeds of the legume, or thesoil surrounding the legume are treated concurrently with the BAP andthe saccharide.
 16. The method of claim 14, wherein the legume, theseeds of the legume, or the soil surrounding the legume are treatedfirst with the BAP and subsequently the legume, the seeds of the legume,or the soil surrounding the legume are treated with the saccharide. 17.The method of claim 14, wherein the legume, the seeds of the legume, orthe soil surrounding the legume are treated first with the saccharideand subsequently the legume, the seeds of the legume, or the soilsurrounding the legume are treated with the BAP.
 18. The method of claim14, comprising administering a composition comprising the BAP to thesoil surrounding the legume at a rate that delivers 10³-10⁵ CFU per gramsoil.
 19. A method for improving growth or health of a plant, the methodcomprising: (a) treating the plant, seeds of the plant, or soilsurrounding the plant with Bacillus amyloliquefaciens subspeciesplantarum (BAP) and (b) treating the plant, the seeds of the plant, orthe soil surrounding the plant with a saccharide comprising pectin or apectin-related saccharide.
 20. A method for improving growth or healthin an animal, the method comprising: (a) administering to the animalBacillus amyloliquefaciens subspecies plantarum and (b) administering tothe animal a saccharide comprising pectin or a pectin-relatedsaccharide.